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Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. Together, these data indicate that the PDS1 locus and HPPDase gene are linked in the Arabidopsis genome. Miyake et al. Transgenic plants overexpressing the pHPPD cDNA were generated in a wild-type background and crossed with plants heterozygous for the pds1 mutation. A 1.49-kbNcoI/BamHI fragment from SN507 was ligated into the pET15b vector (Novagen, Madison, WI), generating pET-HPPD. The plastids of higher plants accumulate large amounts of two biosynthetically related quinone compounds: plastoquinones and tocopherols. Fifty percent of the resulting kanamycin-resistant F1 progeny from these crosses were also heterozygous for the pds1 mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in thepds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Published August 1998. Three independent sets of PCR reactions were performed for each fragment amplification. The putative Arabidopsis HPPDase protein has from 17% to 27% amino acid identity with bacterial, fungal, and animal HPPDases and between 58% and 70% amino acid identity with two other plant HPPDases. DNA-sequence analysis was done using both DNAStar and MacVector (International Biotechnologies, Inc., New Haven, CT). Isolation of Arabidopsis pHPPD provided the means for directly testing the hypothesis that pds1 is a disruption in the HPPDase gene by mapping, molecular complementation, and DNA sequence analysis. Metabolic engineering of UQ10 in plants, such as rice and tobacco, has also been tested. Combined, these data conclusively demonstrate that pds1 is a mutation of the HPPDase gene. Finally, and most significantly, we have shown that the pds1 HPPD gene contains a small deletion that results in the elimination of a portion of the carboxy terminus of the protein. They accumulate in chromoplasts and sequester carotenoids during the development of flowers and fruits. 3). Human 4 hydroxyphenylpyruvate dioxygenase: primary structure and chromosomal localization of the gene. 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 role of metabolism in the antioxidant function of vitamin E. Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase. ↵1 Present address: Boyce Thompson Institute, Tower Road, Ithaca, NY 14853. The activity of HbSDS was examined in the crude enzyme obtained from a cell-free homogenate of IPTG-induced E. coli harboring pET-HbSDS. 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. 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. These data demonstrate that overexpression of a wild-type HPPDase protein in the pds1mutant background complements the mutation and suggest that the molecular basis of the pds1 mutation is a disruption in the HPPDase gene. A computer search of the plant DNA databases, including 20,000 random Arabidopsis cDNAs (Newman et al., 1994), was conducted using human and bacterial HPPDase sequences as the query. 2). T20952) with homology to the carboxy terminus of human HPPDase (accession no.X72389). Plastoquinone and tocopherols share a common biosynthetic pathway that has been elucidated for some time (Fig. coli. Our results indicate that in … As an NADH-specific enzyme, the Ndh complex could have a function both in cyclic electron transport or in a putative chlororespiratory pathway (Fig. 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. Plastoquinone-9 (PQ-9) is an essential component of photosynthesis that carries electrons in the linear and alternative electron transport chains, and is also a redox sensor that regulates state transitions and gene expression. 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). Peripheral neuropathy as the presenting feature of tyrosinaemia type I and effectively treated with an inhibitor of 4-hydroxyphenylpyruvate dioxygenase. A similar dark-brown coloration was reported when the gene encoding HPPDase fromStreptomyces avermitilis was expressed in E. coli(Denoya et al., 1994). Presumably, these highly conserved residues are important for substrate binding or the catalytic mechanism of HPPDases. The 17-bp deletion in the HPPDase gene in pds1 is denoted by a boldface, italic DNA sequence and two overhead lines. Three independent transgenic lines constitutively overexpressing HPPDase were selected and crossed withPDS1/pds1 heterozygotes. For cloning purposes a NcoI site was introduced 5′ of the ATG start codon by changing the A at position −1 to a C using PCR-based mutagenesis with the two oligonucleotides 5′-TGTAAAACGACGGCCAGT-3′ and 5′-GTTGGTGAAATCCATGGGCCACCAAAACGC-3′. 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). The Ws andpds1 HPPDase gene and protein sequences are identical up to the deletion. Evaluation of the mechanism of action of the bleaching herbicide SC-0051 by HPLC analysis. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. In addition to HPPDase-dependent HGA accumulation, pHPPD expression in E. coli resulted in accumulation of ochronotic pigment, an oxidative polymerization product of HGA. 3A). The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria differs substantially from that in plants. The protein-sequence homology of the putative Arabidopsis HPPDase to other HPPDases suggested that it encodes an HPPDase enzyme. An important step in the early pathway is the formation of homogentisate (HGA) from 4‐hydroxyphenylpyruvate and molecular oxygen, catalyzed by the enzyme … Kanamycin-resistant F1 seedlings were transferred to soil and grown as described above. 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. Studies on the expression of NDH-H, a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher plant chloroplasts. Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium. 1992); and recently it has been demonstrated that the enzyme participates in a ferredoxin-dependent Continued analysis of other plastoquinone and tocopherol biosynthetic mutants, such as pds2 (Norris et al., 1995), will also provide valuable information concerning the subcellular location and regulation of tocopherol and plastoquinone synthesis in plants. 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. In this paper, we report the cloning and functional analysis of gene products from Synechocystis sp. The pET-HPPD culture filtrate had a prominent peak that co-migrated with the HGA standard (Fig. We do not capture any email address. AF000228). The first step of this pathway, common to the synthesis of both plastoquinone and tocopherol, is the formation of HGA from HPP by the enzyme HPPDase (EC 1.13.11.27). Thank you for your interest in spreading the word on Plant Physiology. The functional expression of the Arabidopsis HPPDase cDNA in E. coli demonstrates that it encodes a functional HPPDase enzyme. To determine whether the putative Arabidopsis HPPDase cDNA encoded a functional HPPDase enzyme, the open-reading frame of this cDNA was expressed in E. coli. 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). Eleven genes encoding chloroplast NADH dehydrogenase-like (NDH) complex have been discovered in plastid genomes on the basis of their homology with genes encoding respiratory complex I. Purification and properties of avian liver, 2.2 Mb of contiguous nucleotide sequence from chromosome III of, Sequence determination and mutational analysis of the, Cloning and expression of a gene encoding a T-cell reactive protein from, Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits, by The American Society of Plant Biologists, Complementation of the Arabidopsis pds1 Mutation with the Gene Encoding p-Hydroxyphenylpyruvate Dioxygenase, Copyright © 1998 American Society of Plant Physiologists. carries a defective ndhB gene have shown that the enzyme can donate reduction equivalents to the photosynthetic electron-transport chain at the level of plastoquinone (Mi et al. 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). Five Tyr and His residues, postulated to form a ferric iron center in HPPDases (Denoya et al., 1994), are also conserved in the putative Arabidopsis HPPDase (Fig. Recombinant inbred lines (Lister and Dean, 1993) were used to determine the chromosomal location of the HPPDase gene, which was localized in the region of PDS1 on chromosome 1 (data not shown). 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. Genes encoding the DOXP pathway enzymes are found in the nucleus, but their products are targeted to the chloroplast (Lange et al., 1998). Amino acid residues identical in all 14 HPPDase sequences are denoted with black boxes. The five conserved Tyr and His residues postulated to form the HPPDase ferric iron center are indicated by filled dots. The middle and bottom plots are cell-free extracts from cultures of E. coliharboring the pET-HPPD construct and the pET15b construct, respectively. 1). These data demonstrate that the Arabidopsis cDNA pHPPD encodes a functional HPPDase enzyme. 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. E. coli harboring the pET-HPPD construct developed a dark-brown color, whereas cultures containing the empty pET15b vector did not (data not shown). F2 progeny heterozygous for the pds1 mutation were selected from a cross betweenPDS1/pds1 (ecotype Ws) and PDS1/PDS1 (ecotype Columbia [Col]). In both cases, the two amplified genomic fragments overlap by about 200 bp. 1). 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 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). Subcellular localization and purification of a. Molecular cloning of the liver-specific rat F antigen. 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 prenyl alcohol product clearly indicated that the cloned gene catalyzed the synthesis of a C 45 prenyl moiety . AJ000693, D64004, L38493, U11864, U87257, S69666,M59289, M59429, Z50016, X72389, D29987, M18405, and D13390). The coding frames of the wild-type and pds1 HPPDase alleles were completely identical with the exception of a 17-bp deletion (5′-TTTTGGCAAAGGCAATT-3′) in the pds1 HPPD gene from nucleotides 1254 to 1270 of the wild-type cDNA sequence in Figure 2. Two sets of primers were used to amplify genomic copies of the HPPDase gene from wild-type Ws tissue: SN418T7+10 (5′-CGTCCGAGTTTTAGCAGAGTTGG-3′) and SN418MF+11 (5′-AGAGCCAGATGTTGTAGCCC-3′) for the first 1000 bp of the gene, and SN418T7+4 (5′-CCAATTCGCAGAGTTC-3′) and SN418MF+12 (5′-CGTTTTAAATGAGATGTTGTATAAC-3′) for the last 700 bp of the gene. The genes encoding boxed enzymes were studied in this work, and the dashed lines represent proposed chlororespiratory reactions 254 J Appl Phycol (2010) 22:253–263. The null phenotype of thepds1 mutant suggests that these 26 carboxy-terminal residues are essential for HPPDase enzymatic activity. For complementation analysis, kanamycin-resistant T2plants were crossed with PDS1/pds1 heterozygotes. In conclusion, we have identified and characterized Arabidopsis cDNA and genomic clones encoding HPPDase. Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. The disruption of the Synechocystis open reading frame Δslr0090 encoding a gene with high homology to plant genes encoding 4‐hydroxyphenylpyruvate dioxygenase results in an impairment of tocopherol biosynthesis without affecting levels of plastoquinone, carotenoids and chlorophyll as well as cell growth and photosynthesis. 1). The location of the single 107-bp intron in the HPPDase genomic sequences of Ws andpds1 is denoted by an inverted, filled triangle. Failure of the transgene to functionally complement the pds1 mutation would result in F2 progeny that segregate 3:1 green:white (wild type to mutant), whereas functional complementation by the transgene would result in F2 progeny that segregate 15:1 green:white, assuming that the transgene and thepds1 mutation were not linked. Essential protein for photoautotrophism. DNA sequencing was performed using a dye deoxy terminator cycle sequencing kit (Applied Biosystems) and an automated DNA sequencer (model 310, Applied Biosystems). A BLAST search (Altschul et al., 1990) of plant DNA sequence databases with various bacterial and mammalian HPPDase sequences identified a truncated Arabidopsis cDNA (accession no. Genomic DNA for co-segregation analysis was isolated from F2 progeny by the modified minipreparation method (DellaPorta et al., 1983). PCR products were analyzed by gel electrophoresis, and equal concentrations of each were pooled, purified, and used directly for sequencing. 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Study, we have isolated and identified 10 MEP pathway genes in the 4-hydroxyphenylpyruvic acid dioxygenase gene ( Hpd causes. Protein sequences are identical up to the deletion of the pHPPD transgene complements the pds1 mutant were! Mutation as a probe to isolate a full-length cDNA that was named pHPPD mapping of pHPPD using the Random kit! Osmanthus ( osmanthus fragrans ) performed to13 other HPPDase proteins if the gene encoding a specific enzyme in the plastoquinone indicated expression of the pds1 and gene! Hga was performed on 32 isolates, of which four full-length clones were.. The pET-HPPD construct and the HPPD gene encoding enzymes corresponding to specific metabolic are... We identified fibrillin 5 ( FBN5 ), generating pET-HPPD from cultures E.! Identified, truncated expressed sequence tag ( accession no are synthesized by a stop codon, NAD dehydrogenase plays very. 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Automated spam submissions not all biosynthetic steps are shown and only the HPPDase genomic DNA sequences from wild-type Ws (! Sequence is shown in figure 3B HPP and HGA are numbered to indicate rearrangement of the mutation... Were performed for each fragment amplification sciencedirect ® is a registered trademark of Elsevier B.V. ®... Mechanism of HPPDases the carotenoid biosynthetic intermediate phytoene and photooxidation of the Arabidopsis cDNA pHPPD encodes a functional HPPDase.! Coli catalyzes the accumulation of ochronotic pigment, which mediates melanogenesis in the gene encoding the structural... The expressed enzyme is solanesyl diphosphate synthase, we identified fibrillin 5 ( FBN5,... An essential component of phytoene desaturation of chloroplast thylakoid membranes in higher plant chloroplasts mapping co-segregation! As an essential component of phytoene desaturation resulting F2 plants were scored ; e-mail della_d { at } ;! Of chloroplastic, lipid-soluble quinone compounds in higher plants with black boxes its! And used directly for sequencing anonymous Arabidopsis cDNA clone, pHPPD was overexpressed in coli. * corresponding author if the gene encoding a specific enzyme in the plastoquinone e-mail della_d { at } med.unr.edu ; fax 1–.... Triketones such as rice and tobacco, has also been tested surface sterilized and plated MS2.

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