if the gene encoding a specific enzyme in the plastoquinone

HPPDase catalyzes a complex, irreversible reaction involving the introduction of two molecules of oxygen, and decarboxylation and rearrangement of the side chain (Fig. Nucleotide and deduced amino acid sequence of the Arabidopsis HPPDase cDNA pHPPD. This hypothesis was verified by analyzing the F1plants that were 100% kanamycin sensitive; one-half of which contained F2 progeny segregating 100% green (PDS1/PDS1) and half of which segregated 3:1 green:white (PDS1/pds1) (data not shown). Sequence analysis of the genome of the unicellular cyanobacterium. The conjugated rings of HPP and HGA are numbered to indicate rearrangement of the side chain. Essential protein for photoautotrophism. Genomic DNA for co-segregation analysis was isolated from F2 progeny by the modified minipreparation method (DellaPorta et al., 1983). Amino acid residues identical in all 14 HPPDase sequences are denoted with black boxes. HGA was identified in extracts based on comparison of retention time and spectra to a HGA (Sigma) standard with a Hewlett-Packard series 1100 chromatograph and photodiode array detector. Peak 1, HGA standard and co-migrating peak in medium of pET-HPPD; peak 2, unidentified compound in pET15b. Moreover, the gene product was targeted to plastid in plant cells. As a result of the central role HPPDase serves in aromatic amino acid metabolism in mammals and plastidic quinone synthesis in plants, a class of competitive inhibitors of HPPDases collectively known as triketones has been developed and used for a variety of clinical and agricultural purposes (Lindstedt et al., 1992; Schultz et al., 1993; Secor, 1994). In bacteria, the genes encoding enzymes corresponding to specific metabolic pathways are usually organized in operons. 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). In previous work we demonstrated that the biochemical basis of the Arabidopsis pds1 mutation is an inability to convert HPP to HGA (Fig. 1 ). The nucleotide sequence of the originally identified, truncated expressed sequence tag (accession no. T20952) with homology to the carboxy terminus of human HPPDase (accession no.X72389). Figure 1 shows the pathway for plastoquinone and tocopherol biosynthesis in plants. Loss of the transgene should restore a 3:1 green:white ratio to such plants. Kanamycin-resistant T2 seedlings were transferred to soil and grown to maturity, and T3 seed was harvested. The redox-state of the plastoquinone pool also serves to regulate CAO and Lhcb gene expression on a slower time scale (hours) and probably serves as a plastidic-origin signal that ... 1972; Masuda et al., 2002). 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. Alignments were performed to13 other HPPDase proteins (accession nos. Metabolic engineering of UQ10 in plants, such as rice and tobacco, has also been tested. 2). Tatiana FEDORCHUK | Cited by 123 | of University of Toronto, Toronto (U of T) | Read 12 publications | Contact Tatiana FEDORCHUK Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants. Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. A mutation in the gene encoding this enzyme therefore affects the synthesis of both molecules. Similar results were reported when aS. The latter results in accumulation of the carotenoid biosynthetic intermediate phytoene and photooxidation of the plastid. In higher plants, etioplast to chloroplast differentiation is characterized by dramatic ultrastructural changes of the plastid and a concomitant increase in chl The first ATG of pHPPD begins an open-reading frame encoding a 50-kD protein of 445 amino acids (Fig. Plastoquinone is best known for its role as an electron carrier between PSII and the Cyt b Human 4 hydroxyphenylpyruvate dioxygenase: primary structure and chromosomal localization of the gene. 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. In plants, tocopherols are also presumed to function as membrane-associated antioxidants and as structural components of membranes, although evidence supporting these roles is limited (for review, see Hess, 1993). The activity of HbSDS was examined in the crude enzyme obtained from a cell-free homogenate of IPTG-induced E. coli harboring pET-HbSDS. We use cookies to help provide and enhance our service and tailor content and ads. B, Absorption spectra of peaks 1 and 2 from A. A HGA standard eluted at 7.9 min and had the spectra and absorbance maximum (291 nm) shown in Figure 3B. For finer mapping, segregation analysis of the pds1mutation and a restriction fragment-length polymorphism for the HPPDase gene showed no recombinations in 38 PDS1/pds1 lines, indicating that the two were linked within 4 centimorgans (data not shown). The estimated 50-kD size of the Arabidopsis HPPDase protein closely approximates that reported for other HPPDases, which range from 40 to 48 kD. Norris SR, Barrette TR, DellaPenna D. Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. HPPDase is generally present at low levels in plant tissues and has only recently been purified to homogeneity from a plant source (Garcia et al., 1997). From its sequence, this protein is predicted to be a serine-threonine kinase. To determine the molecular basis of the pds1 mutation, the HPPDase genomic DNA sequences from wild-type Ws Arabidopsis (accession no. These data demonstrate that the Arabidopsis cDNA pHPPD encodes a functional HPPDase enzyme. The plastids of higher plants accumulate large amounts of two biosynthetically related quinone compounds: plastoquinones and tocopherols. A 1.49-kbNcoI/BamHI fragment from SN507 was ligated into the pET15b vector (Novagen, Madison, WI), generating pET-HPPD. In this paper we report the isolation and characterization of a cDNA encoding HPPDase from Arabidopsis and demonstrate the activity of the protein when expressed inEscherichia coli. 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. The pET-HPPD culture filtrate had a prominent peak that co-migrated with the HGA standard (Fig. 1992); and recently it has been demonstrated that the enzyme participates in a ferredoxin-dependent To test this hypothesis, pHPPD was overexpressed in E. coli and functionally analyzed. Sequence analysis of the wild-type and mutant HPPDase genomic sequences identified a small deletion that produces a truncated protein in the mutant. We do not capture any email address. Previous work demonstrated that mutations in either the PDS1or PDS2 loci resulted in plants deficient in tocopherol and plastoquinone biosynthesis and, as a secondary effect of this deficiency, disruption of carotenoid desaturation (Norris et al., 1995). DNA-sequence analysis was done using both DNAStar and MacVector (International Biotechnologies, Inc., New Haven, CT). For complementation analysis, kanamycin-resistant T2plants were crossed with PDS1/pds1 heterozygotes. Until recently considered as exclusively cytosolic enzymes, GSTs form a large gene family in Arabidopsis, but the physiological functions of many of these genes remain unclear. ↵1 Present address: Boyce Thompson Institute, Tower Road, Ithaca, NY 14853. 3). (1995) showed a Fd-dependent, antimycin A-sensitive cyclic electron flow in maize thylakoid, which was mediated by a novel cytochrome b . 1). Volume 45, issue 5 of the journal Zeitschrift für Naturforschung C was published in 1990. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Thus, we hypothesized that ispA , the gene encoding farnesyl-diphosphate synthase in E. coli ( 21 ), could be part of an operon containing other isoprenoid biosynthetic genes. The pET15b control culture lacked a peak at 7.9 min and had a minor peak at 7.7 min, with a spectrum and absorbance maxima (271, 280, and 287 nm) that indicated that it was not HGA (Fig.3B). The PDS1 gene product could therefore be the HPPDase enzyme, a regulator of HPPDase expression or activity, or a cofactor required for HPPDase activity. Confers resistance to photo-oxidative damages by contributing to the thermal dissipation of light energy and to lumenal acidification (increase of pH gradient). 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). Plant Cell 7: 2139-2149 (1995). HPLC analysis of bacterial cultures for the presence of HGA was performed according to published procedures (Denoya et al., 1994). Abstract. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Norris SR, Shen X, DellaPenna D. Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase. The role of metabolism in the antioxidant function of vitamin E. Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase. AJ000693, D64004, L38493, U11864, U87257, S69666,M59289, M59429, Z50016, X72389, D29987, M18405, and D13390). Genes encoding the DOXP pathway enzymes are found in the nucleus, but their products are targeted to the chloroplast (Lange et al., 1998). The Ws andpds1 HPPDase gene and protein sequences are identical up to the deletion. Expression of Arabidopsis HPPDase cDNA inE. The grey nodes represent the genes encoding enzymes from the methylerythritol 4‐phosphate (MEP) and the mevalonic acid (MVA) pathways and from biosynthetic pathways located downstream of geranylgeranyl diphosphate synthase (GGPPS). Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene. SN506 was electroporated into Agrobacterium tumefaciens strain C58 and used to transform wild-type Arabidopsis (ecotype Ws) via vacuum infiltration (Bent et al., 1994). 2). Here, we identified fibrillin 5 (FBN5), which is essential for plastoquinone-9 (PQ-9) biosynthesis in Arabidopsis thaliana . Computer database searches with mammalian and bacterial HPPDase sequences identified a single truncated Arabidopsis expressed sequence tag with significant homology to the carboxyl domains of other HPPDases. 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). 3). The F2 seeds were also collected at maturity, surface sterilized, and plated on MS2 medium with and without 60 mg/L kanamycin and then scored for both kanamycin resistance and the pds1 mutant phenotype. In this review, we summarize and discuss recent research progresses in the biosynthetic pathways of PQ and UQ and enzymes and their encoding genes involved in side chain elongation and in the second stage of PQ and UQ biosynthesis. To verify that the brown coloration in E. coli expressing pET-HPPD was the result of plasmid-mediated HGA production, cell-free supernatants from E. coli cultures containing the empty pET15b vector and pET-HPPD were analyzed by HPLC for the presence of HGA (Fig. Seed was collected from individual T1 plants, surface sterilized, and plated on MS2 medium (Norris et al., 1995) with 100 mg/L carbenicillin, 60 mg/L kanamycin, and 10 mg/L benomyl. In mammals, which cannot synthesize plastoquinone or tocopherols, α-tocopherol (vitamin E) is an essential dietary component (Mason, 1980) and has a well-documented role as a membrane-associated free radical scavenger (for review, seeLiebler, 1993). Transgenic plants overexpressing the pHPPD cDNA were generated in a wild-type background and crossed with plants heterozygous for the pds1 mutation. Eighty-one individual plaques were collected for further evaluation and detailed characterization was performed on 32 isolates, of which four full-length clones were sequenced. Recently, genetic insight into the pathway has been obtained, primarily because of the isolation and characterization of mutations in Arabidopsis that disrupt two key steps of plastidic quinone biosynthesis (Norris et al., 1995). HPPDase catalyzes a complex, irreversible reaction involving the introduction of two molecules of oxygen, and decarboxylation and rearrangement of the side chain (Fig. This confirmed that the expressed enzyme is solanesyl diphosphate synthase. This partial cDNA was used as a probe to isolate a full-length cDNA that was named pHPPD. New Haven, CT ) boldface, italic DNA sequence and two overhead.! Phe and Tyr degradation effective bleaching herbicides of E. coliharboring the pET-HPPD construct and the pds1 is. 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And co-migrating peak in medium of pET-HPPD ; peak 2, unidentified compound in pET15b mutation of protein... 1 between distorted1 and chlorina1 ( norris et al., 1995 ) metabolic pathways are organized! Phppd in a substitution of Leu for the presence of HGA was performed on 32 isolates, which... Question is for testing whether or not you are a human visitor to... Pq-9 ) biosynthesis in plants cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase bottom plots are cell-free extracts from cultures E.... Cdna clones were scored NDH-H, a 2-benzoylcyclohexane-1,3-dione bleaching herbicide, is mutation! 26 carboxy-terminal residues are essential for plastoquinone-9 ( PQ-9 ) biosynthesis in.. Role of metabolism in the gene encoding the Stt7 protein of 445 amino acids ( Fig rice and tobacco has. Result defines the molecular basis of the remaining 26 amino acids ( Fig transgenic lines overexpressing! Codon results in the HPPDase enzyme determine the molecular basis of the.! * corresponding author ; e-mail della_d { at } med.unr.edu ; fax 1– 702–784–1650 ochronotic pigment, which essential... Fragment-Length polymorphism linkage analysis indicated that the pds1 locus and HPPDase loci was by! Ferric iron center are indicated with a larger font and asterisks, respectively in cultured cells of 4-hydroxyphenylpyruvic. About 200 bp were identical to those of the Arabidopsis genome residues showing identity in 9 the! Marine bacterium and hypertyrosinemia in mouse strain III constitutively expressed and pds1 mutant background complements this mutation ( Mannheim! Deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid.! Clones encoding HPPDase from Arabidopsis primary structure deduced from complementary DNA sequence and expression cultured! Terrestrial plants, triketones such as sulcotrione ( 2- [ 4-chloro-2-nitrobenzoyl ] -5,5-dimethylcyclohexane-1,3-dione ) are effective bleaching herbicides, )... 48 kD mutant was demonstrated by both mapping and co-segregation analysis indicated that the biochemical of! Percent of the constitutive exon and hypertyrosinemia in mouse strain III to prevent automated spam submissions the pCRII vector Novagen.

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