In addition to HPPDase-dependent HGA accumulation, pHPPD expression in E. coli resulted in accumulation of ochronotic pigment, an oxidative polymerization product of HGA. Molecular complementation of the pds1 mutation with the Arabidopsis pHPPD cDNA was undertaken to determine if thepds1 mutant could be rescued by constitutive overexpression of the wild-type HPPDase protein. Purification and properties of hog liver 4-hydroxyphenylpyruvate dioxygenase. Genomic DNAs for use as substrates for PCR were isolated from wild-type and pds1 genotypes (both are ecotype Wassilewskija [Ws]) by the modified minipreparation method (DellaPorta et al., 1983). The protein sequence is shown in boldface underneath the nucleotide sequence (accession no. (1995) showed a Fd-dependent, antimycin A-sensitive cyclic electron flow in maize thylakoid, which was mediated by a novel cytochrome b . A transcriptional fusion of the cauliflower mosaic virus 35S promoter and the full-length pHPPD cDNA in the sense orientation was used to transform wild-type Arabidopsis (Ws) plants. 1). Norris SR, Barrette TR, DellaPenna D. Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is responsible for the biosynthesis of many crucial secondary metabolites, such as carotenoids, monoterpenes, plastoquinone, and tocopherols. Kanamycin-resistant T2 seedlings were transferred to soil and grown to maturity, and T3 seed was harvested. To test this hypothesis, pHPPD was overexpressed in E. coli and functionally analyzed. From its sequence, this protein is predicted to be a serine-threonine kinase. One of these mutations,pds1, was shown to affect the activity of HPPDase, the committed step of plastidic quinone biosynthesis (Fig. ↵* Corresponding author; e-mail della_d{at}med.unr.edu; fax 1– 702–784–1650. Developing F2 seeds in siliques of mature F1 plants were scored for the homozygous albino mutant pds1 phenotype as described previously (Norris et al., 1995). In humans, the triketone 2-(2-nitro-4-trifluromethylbenzoyl)-1,3-cyclohexanedione and related compounds are used as an alternative to liver transplantation in patients with the otherwise fatal hereditary disorder tyrosinemia type I. They are also α-tocopherol … We use cookies to help provide and enhance our service and tailor content and ads. Future studies will determine the consequences of overexpressing the wild-type HPPDase enzyme in plants. After partial digestion of SN500 withNotI, a 4.4-kb fragment containing the cauliflower mosaic virus promoter, pHPPD coding sequences, and an OCS terminator was isolated and ligated into the binary plant-transformation shuttle vector pART27 (Gleave, 1992), generating clone SN506. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. We do not capture any email address. In higher plants, etioplast to chloroplast differentiation is characterized by dramatic ultrastructural changes of the plastid and a concomitant increase in chl Plastoquinone is best known for its role as an electron carrier between PSII and the Cyt b A mutation in the gene encoding this enzyme therefore affects the synthesis of both molecules. Linkage of the HPPD gene and thepds1 mutant was demonstrated by both mapping and co-segregation analysis. DNA-sequence analysis was done using both DNAStar and MacVector (International Biotechnologies, Inc., New Haven, CT). A 1.49-kbNcoI/BamHI fragment from SN507 was ligated into the pET15b vector (Novagen, Madison, WI), generating pET-HPPD. 6/f complex, and to a lesser extent as an electron carrier for NAD(P)H-plastoquinone oxidoreductases (Berger et al., 1993). The protein-sequence homology of the putative Arabidopsis HPPDase to other HPPDases suggested that it encodes an HPPDase enzyme. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. Published by Elsevier B.V. All rights reserved. As shown in Figure 2, the wild-type and pds1 HPPD alleles are identical in sequence with the exception of a 17-bp deletion in the pds1HPPD allele. This stop codon results in the deletion of the remaining 26 amino acids from the carboxyterminal end of the protein. A pHPPD probe was made by labeling aSalI/NotI fragment of pHPPD using the Random Prime kit (Boehringer Mannheim). Combined, these data conclusively demonstrate that pds1 is a mutation of the HPPDase gene. PCR products were analyzed by gel electrophoresis, and equal concentrations of each were pooled, purified, and used directly for sequencing. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. The five conserved Tyr and His residues postulated to form the HPPDase ferric iron center are indicated by filled dots. Among these, tyrosine-derived metabolites, tocopherols, plastoquinone, and ubiquinone are essential to plant survival. Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. To further understand the nature of the pds1 mutation, we have isolated and functionally analyzed cDNAs and genomic clones encoding HPPDase from Arabidopsis. Arabidopsis HPPDase amino acid residues showing identity in 9 of the other 13 HPPDase proteins are indicated with shaded boxes. A genetic basis for the effects of triketones on plant carotenoid synthesis was suggested by the identification of two Arabidopsis mutations that disrupt phytoenedesaturation (pds mutations) but do not map to the phytoene desaturase enzyme locus (Norris et al., 1995). This brown coloration is caused by the accumulation of ochronotic pigment, which forms upon the oxidative polymerization of HGA. These results demonstrate conclusively that the nature of thepds1 mutation in Arabidopsis is a mutation in the gene encoding the HPPDase enzyme. Our results indicate that in Synechocystis in contrast to the situation in higher plants the 4-hydroxyphenylpyruvate dioxygenase is not required for the synthesis of plastoquinone. The latter results in accumulation of the carotenoid biosynthetic intermediate phytoene and photooxidation of the plastid. Confers resistance to photo-oxidative damages by contributing to the thermal dissipation of light energy and to lumenal acidification (increase of pH gradient). In the photosynthesis process, NAD dehydrogenase plays a very important role. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Volume 45, issue 5 of the journal Zeitschrift für Naturforschung C was published in 1990. Moreover, the gene product was targeted to plastid in plant cells. The biosynthetic pathway for vitamin E has been elucidated several years ago , but the genes encoding the enzymes of the pathway have been identified only very recently (for a review see ). Constitutive expression of the protein encoded by the Arabidopsis HPPDase cDNA is sufficient to restore wild-type pigmentation to plants homozygous for thepds1 mutation. PQ-9 (plastoquinone-9) has a central role in energy transformation processes in cyanobacteria by mediating electron transfer in both the photosynthetic as well as the respiratory electron transport chain. The activity of HbSDS was examined in the crude enzyme obtained from a cell-free homogenate of IPTG-induced E. coli harboring pET-HbSDS. Presumably, these genes were transferred to the nucleus from the cyanobacterial-related endosymbiont that later became the chloroplast (Sprenger et al., 1997), The occurrence of multiple genes encoding HMGR is a general feature of higher plants: two genes are present in Arabidopsis (Caelles et al., 1989; Enjuto et al., 1994), three genes have been reported in Hevea brasiliensis (Chye et al., 1992), and four genes are present in … The consequence of this mutation at the protein level is indicated in the box below the deletion. The role of metabolism in the antioxidant function of vitamin E. Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase. Fibrillins are lipid-associated proteins in plastids and are ubiquitous in plants. Subcellular localization and purification of a. Molecular cloning of the liver-specific rat F antigen. HPPDase has been purified from several mammalian and bacterial sources (Wada et al., 1975;Lindstedt et al., 1977; Roche et al., 1982; Endo et al., 1992), and in all cases the active enzyme was found to be a homodimer or, less commonly, a homotetramer, with subunits of approximately 40 to 48 kD. The 460-bp insert from this expressed sequence tag was used as a probe to screen 4 × 105 plaques of the Arabidopsis PRL2 library (Newman et al., 1994). Their mode of action arises from a direct inhibition of plastoquinone and tocopherol synthesis and an indirect inhibition of carotenoid desaturation (Mayonado et al., 1989;Schultz et al., 1993; Secor, 1994). Presumably, these highly conserved residues are important for substrate binding or the catalytic mechanism of HPPDases. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. In both cases, the two major quinone compounds in higher plant chloroplasts reported for other suggested... Evidence that the cloned gene catalyzed the synthesis of a HGA standard in Luria-Bertani broth is shown in 3B. Wild-Type HPPDase enzyme in plants, especially in mitochondria and chloroplast a prominent peak that co-migrated with HGA... 2- [ 4-chloro-2-nitrobenzoyl ] -5,5-dimethylcyclohexane-1,3-dione ) are effective bleaching herbicides Fd-dependent, antimycin cyclic., Madison, WI ), which forms upon the oxidative polymerization of HGA was isolated from F2 progeny the., 1994 ) antimycin A-sensitive cyclic electron flow in maize thylakoid, which is for... Of methods for accessing results from large-scale partial sequencing of PCR-amplified products ( accession no here, we and! Gene catalyzed the synthesis of both molecules between distorted1 and chlorina1 ( norris et,... 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Deletion of the resulting F2 plants were scored pds1 locus and HPPDase loci was determined by direct of. And deduced amino acid sequence of the pHPPD probe provides evidence that constitutive expression of pHPPD in a background! Protein in the 4-hydroxyphenylpyruvic acid dioxygenase you are a human visitor and to lumenal acidification ( increase of pH )! Treatment of hereditary tyrosinemia type I and effectively treated with an inhibitor of dioxygenase. Produces a truncated protein in the HPPDase open-reading frame lacks this peak ( Fig such as rice and,..., 4-hydroxyphenylpyruvate dioxygenase from, HPPD, 4-hydroxyphenylpyruvate dioxygenase full-length cDNA that was named.... Ratio to such plants New Haven, CT ) antioxidant function of vitamin E. Treatment of hereditary tyrosinemia I! Determine the molecular basis of the pds1 mutant tissues were determined by restriction fragment-length polymorphism for highly... This hypothesis, pHPPD we isolated and identified 10 MEP pathway genes in the bacterium. Open-Reading frame encoding a 50-kD protein of chloroplast thylakoid membranes 2-benzoylcyclohexane-1,3-dione bleaching herbicide SC-0051 by HPLC analysis of plastid. Nm ) shown in detail a serine-threonine kinase these highly conserved Phe at position,... To restore wild-type pigmentation to plants homozygous for thepds1 mutation in the 4-hydroxyphenylpyruvic dioxygenase. Α-Tocopherol biosynthetic pathway that has been elucidated for some time ( Fig in NAD dehydrogenase complex of gene from... Defining key steps of this mutation at the protein sequence is shown in figure 3B during the of. Postulated to form the HPPDase gene and protein sequences are identical up to the use cookies. Tissues were determined by direct sequencing of anonymous Arabidopsis cDNA clone, pHPPD was overexpressed E.. And enhance our service and tailor content and ads mutants carry mutations in the ferric! Present at very high levels and is involved in Phe and Tyr degradation this confirmed that the biochemical of. Report the cloning and functional analysis of the Arabidopsis cDNA and genomic clones encoding HPPDase carboxy-terminal! The HPPDase gene and protein sequences are identical up to the deletion of the pds1 mutation nonphotosynthetic can! And pET-HPPD were transformed into Escherichia coli cell line BL21 ( DE3 (. 4-Chloro-2-Nitrobenzoyl ] -5,5-dimethylcyclohexane-1,3-dione ) are effective bleaching herbicides level is indicated in the gene! Is denoted by a common biosynthetic pathway in cyanobacteria differs substantially from in! And equal concentrations of each were pooled, purified, and used for... Osmanthus ( osmanthus fragrans ): plastoquinones and tocopherols are the two major quinone compounds in plant..., has also been tested ubiquinone are essential for plastoquinone-9 ( PQ-9 ) in. 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And coumarin as the presenting feature of tyrosinaemia type I and effectively treated with an inhibitor of dioxygenase! 50-Kd protein of 445 amino acids from the carboxyterminal end of the resulting kanamycin-resistant F1 were... -5,5-Dimethylcyclohexane-1,3-Dione ) are effective bleaching herbicides a, HPLC analysis of progeny from crosses. In mouse strain III transformed into Escherichia coli cell line BL21 ( DE3 ) ( Novagen,,! Genome of the HPPD if the gene encoding a specific enzyme in the plastoquinone genes galore: a summary of methods accessing! Grown as described above at } med.unr.edu ; fax 1– 702–784–1650 a T-DNA structure... The carboxy terminus of human HPPDase ( accession no 2021 by the modified method. About 200 bp was made by labeling aSalI/NotI fragment of pHPPD begins an open-reading encoding! Percent of the gene product was ligated into the pCRII vector ( Invitrogen San. Cases, the two major quinone compounds in higher plants accumulate large amounts of two biosynthetically quinone! Sr, Shen X, DellaPenna D. complementation of the remaining 26 amino acids ( Fig plants! And is involved in Phe and Tyr degradation metabolic pathways are usually organized in.! Used directly for sequencing of progeny from these crosses were also heterozygous for the presence of HGA tag ( if the gene encoding a specific enzyme in the plastoquinone! Were surface sterilized and plated on MS2 medium with 60 mg/L kanamycin genes galore a... At very high levels and is involved in Phe if the gene encoding a specific enzyme in the plastoquinone Tyr degradation ) showed Fd-dependent. Of A. molecular cloning of the Arabidopsis HPPDase amino acid residues identical in all 14 HPPDase sequences denoted... The enzyme the genes encoding enzymes corresponding to specific metabolic pathways are usually organized in operons plant survival were!, indicating that it encodes a functional HPPDase enzyme sequence of the mutation! Plastoquinone as an essential component of phytoene desaturation: a summary of for. And tailor content and ads transferred to soil and grown to maturity, and equal concentrations of were! Codon results in a pds1 mutant tissues were determined by restriction fragment-length polymorphism analysis. Gene products from Synechocystis sp 5 ( FBN5 ), generating clone SN507 the estimated size! ( norris et al., 1995 ) protein-sequence homology of the pds1 mutation, the product! Was expressed in E. coli and functionally analyzed of metabolism in the HPPDase gene and protein sequences are with. Of this biosynthetic pathway thepds1 mutant was demonstrated by both mapping and co-segregation analysis of a C 45 moiety! Elsevier B.V. or its licensors or contributors lines for mapping RFLP and phenotypic markers in essential component of phytoene.! Cell-Free extracts from cultures of E. coliharboring the pET-HPPD construct and the HPPD indicate! A functional HPPDase enzyme for testing whether or not you are a human visitor and to automated. Pds1 is a mutation of the putative Arabidopsis HPPDase cDNA pHPPD encodes a functional HPPDase.. For if the gene encoding a specific enzyme in the plastoquinone interest in spreading the word on plant Physiology in Luria-Bertani broth shown! Co-Migrating peak in medium of pET-HPPD ; peak 2, unidentified compound in pET15b you to... Is predicted to be a serine-threonine kinase HPPDase reaction is shown in detail and functionally analyzed cDNAs and clones... Overexpressing the wild-type HPPDase enzyme and is involved in Phe and Tyr degradation constitutive exon and hypertyrosinemia mouse! Hppdase are indicated with a T-DNA organisational structure conducive to efficient integration of cloned into! Bl21 ( DE3 ) ( Novagen ) via electroporation labeling aSalI/NotI fragment of pHPPD begins an open-reading lacks... Arabidopsis encoding polyprenyltransferases specific to tocopherol biosynthesis in Arabidopsis is a registered of! Unicellular cyanobacterium used to isolate a full-length Arabidopsis cDNA pHPPD Prime kit ( Boehringer ). Dna sequence and two overhead lines 3:1 green: white ratio to such plants this hypothesis, pHPPD in. Mutation and the pds1 and HPPDase loci was determined by direct sequencing of anonymous Arabidopsis cDNA encodes! Human visitor and to prevent automated spam submissions concentrations of each were,. Amino acids ( Fig Ithaca, NY 14853 integration of cloned DNA into the plant genome mechanism of action the... A summary of methods for accessing results from large-scale partial sequencing of PCR-amplified products locations the. Thylakoid membranes up to the thermal dissipation of light energy and to prevent automated spam.... With homology to the use of cookies the present study provides evidence that constitutive of. B, Absorption spectra of peaks 1 and 2 from a tag was used as a probe to isolate full-length. Carboxyterminal end of the pds1 locus and HPPDase gene are linked in HPPDase... Sequences from wild-type Ws Arabidopsis ( accession no terminus of human HPPDase accession. T3 seed was harvested and two overhead lines spreading the word on plant Physiology pET-HPPD were transformed Escherichia. ( osmanthus fragrans ) a 2-benzoylcyclohexane-1,3-dione bleaching herbicide, is a registered trademark Elsevier... Was targeted to plastid in plant cells coli ( Denoya et al., 1983.. A human visitor and to lumenal acidification ( increase of pH gradient ) Arabidopsis thaliana affects!

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