if the gene encoding a specific enzyme in the plastoquinone

On the basis of the results presented herein, we conclude that the stromal reductant is NADH and that the Ndh complex functions as an NADH:plastoquinone oxidoreductase. This confirmed that the expressed enzyme is solanesyl diphosphate synthase. Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. In addition to HPPDase-dependent HGA accumulation, pHPPD expression in E. coli resulted in accumulation of ochronotic pigment, an oxidative polymerization product of HGA. For complementation analysis, kanamycin-resistant T2plants were crossed with PDS1/pds1 heterozygotes. Digestion of Ws and Col genomic DNA withNcoI gave a restriction fragment-length polymorphism for the pHPPD probe. E. coli containing a control plasmid without the HPPDase open-reading frame lacks this peak (Fig. In this paper, we report the cloning and functional analysis of gene products from Synechocystis sp. Subcellular localization and purification of a. Molecular cloning of the liver-specific rat F antigen. NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. Presumably, these highly conserved residues are important for substrate binding or the catalytic mechanism of HPPDases. Peak 1, HGA standard and co-migrating peak in medium of pET-HPPD; peak 2, unidentified compound in pET15b. 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. Fibrillins are lipid-associated proteins in plastids and are ubiquitous in plants. Here, we identified fibrillin 5 (FBN5), which is essential for plastoquinone-9 (PQ-9) biosynthesis in Arabidopsis thaliana . 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. Three complementary approaches were undertaken to determine whether the gene identified by the pds1 mutation encodes HPPDase: co-segregation of the pds1 mutation and HPPD gene, functional complementation of the pds1 mutant with the wild-type pHPPD cDNA, and DNA-sequence analysis of the wild-type and mutant HPPD alleles. 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. 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). The amplified product was ligated into the pCRII vector (Invitrogen, San Diego, CA), generating clone SN507. Plastoquinone and tocopherols share a common biosynthetic pathway that has been elucidated for some time (Fig. 1 ). 2). Although it was clear from previous work that the pds1mutation affected HPPDase activity, we could not determine whether thepds1 mutation directly or indirectly affected the HPPDase enzyme (Norris et al., 1995). avermitilis HPPDase was expressed in E. coli(Denoya et al., 1994). Thank you for your interest in spreading the word on Plant Physiology. 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). Three independent transgenic lines constitutively overexpressing HPPDase were selected and crossed withPDS1/pds1 heterozygotes. Isolate 18 was chosen for further studies and renamed pHPPD. Human 4 hydroxyphenylpyruvate dioxygenase: primary structure and chromosomal localization of the gene. Copyright © 2002 Federation of European Biochemical Societies. Among these, tyrosine-derived metabolites, tocopherols, plastoquinone, and ubiquinone are essential to plant survival. 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). As discussed previously, Arabidopsis plants homozygous for thepds1 mutation are unable to synthesize both plastoquinone and tocopherols because of an inability to convert HPP to HGA (Fig. 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). We use cookies to help provide and enhance our service and tailor content and ads. The tyrosine metabolism pathway serves as a starting point for the production of a variety of structurally diverse natural compounds in plants, such as tocopherols, plastoquinone, ubiquinone, betalains, salidroside, benzylisoquinoline alkaloids, and so on. Volume 45, issue 5 of the journal Zeitschrift für Naturforschung C was published in 1990. 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). 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). 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. As an NADH-specific enzyme, the Ndh complex could have a function both in cyclic electron transport or in a putative chlororespiratory pathway (Fig. Published August 1998. Confers resistance to photo-oxidative damages by contributing to the thermal dissipation of light energy and to lumenal acidification (increase of pH gradient). The consequence of this deletion at the protein level is the loss of 26 carboxy-terminal amino acids from the HPPDase protein, including a tight cluster of several amino acids that are conserved in all HPPDase proteins in the database. 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. 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. The resulting F1 seeds were surface sterilized and plated on MS2 medium with 60 mg/L kanamycin. 1). 1). Kanamycin-resistant F1 plants were selected and selfed, and the resulting F2 plants were scored. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. et al., 1998). This deletion results in a substitution of Leu for the highly conserved Phe at position 419, followed immediately by a stop codon. By continuing you agree to the use of cookies. This stop codon results in the deletion of the remaining 26 amino acids from the carboxyterminal end of the protein. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. 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. 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. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Similar results were reported when aS. The estimated 50-kD size of the Arabidopsis HPPDase protein closely approximates that reported for other HPPDases, which range from 40 to 48 kD. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. These kanamycin-resistant, pds1 heterozygous F1 plants were then selfed, and segregation of their F2 progeny for both kanamycin resistance and the pds1 phenotype was determined (TableI). 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). The middle and bottom plots are cell-free extracts from cultures of E. coliharboring the pET-HPPD construct and the pET15b construct, respectively. Comparison of the putative Arabidopsis HPPDase protein sequence with HPPDase protein sequences from 13 other diverse species identified 37 conserved residues clustered primarily in the carboxy region of the protein (Fig. The digested DNA was separated on a 0.6% agarose gel and transferred to a nylon membrane (Micron Separations, Westborough, MA). of chlorophyll biosynthesis enzymes and that of the Lhcb genes in the model organism Dunaliella salina. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. The role of metabolism in the antioxidant function of vitamin E. Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase. Despite this structural similarity, chloroplast NDH and its evolutionary origin NDH-1 in cyanobacteria accept elec … Inhibition of barnyardgrass 4-hydroxyphenylpyruvate dioxygenase by sulcotrione. To further understand the nature of the pds1 mutation, we have isolated and functionally analyzed cDNAs and genomic clones encoding HPPDase from Arabidopsis. Loss of the transgene should restore a 3:1 green:white ratio to such plants. The CO2 lost and molecular oxygen introduced by HPPDase are indicated with a larger font and asterisks, respectively. This gene encodes a [PT][1] that differs from other known [PT][1]s, including flavonoid-specific [PT][1]s, in polypeptide sequence. However, despite this compelling evidence, it could not be determined whether the pds1 mutation directly or indirectly affected the HPPDase enzyme (Norris et al., 1995). 1992); and recently it has been demonstrated that the enzyme participates in a ferredoxin-dependent As shown in Figure 3, E. coli cultures expressing pHPPD accumulate a compound that co-migrates with, and has a spectrum identical to, the HGA standard (Fig. An important step in the early pathway is the formation of homogentisate (HGA) from 4‐hydroxyphenylpyruvate and molecular oxygen, catalyzed by the enzyme … The Ws andpds1 HPPDase gene and protein sequences are identical up to the deletion. 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. The protein-sequence homology of the putative Arabidopsis HPPDase to other HPPDases suggested that it encodes an HPPDase enzyme. A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III. E. coli harboring the pET-HPPD construct developed a dark-brown color, whereas cultures containing the empty pET15b vector did not (data not shown). FQR is a putative enzyme that reduces plastoquinone using Fd as a direct electron donor and has been characterized as a binding site for antimycin A, a specific inhibitor of cyclic electron flow. In plants, triketones such as sulcotrione (2-[4-chloro-2-nitrobenzoyl]-5,5-dimethylcyclohexane-1,3-dione) are effective bleaching herbicides. SN506 was electroporated into Agrobacterium tumefaciens strain C58 and used to transform wild-type Arabidopsis (ecotype Ws) via vacuum infiltration (Bent et al., 1994). AF000228). The conjugated rings of HPP and HGA are numbered to indicate rearrangement of the side chain. This deletion causes a frame shift and substitution of a Leu for the conserved Phe at position 419, followed immediately by a stop codon (Fig. 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). 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. 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. Norris SR, Shen X, DellaPenna D. Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase. AF060481) and pds1 mutant tissues were determined by direct sequencing of PCR-amplified products. Identified, truncated expressed sequence tag was used as a probe to isolate a full-length cDNA that named..., a 2-benzoylcyclohexane-1,3-dione bleaching herbicide SC-0051 by HPLC analysis of the side chain also …! Which four full-length clones were sequenced the unicellular cyanobacterium, and ubiquinone are essential for plastoquinone-9 ( PQ-9 ) in! Which is essential for HPPDase enzymatic activity dna-sequence analysis was done using both DNAStar MacVector! Were analyzed by gel electrophoresis, and the HPPD gene and thepds1 mutant suggests that 26! Hypertyrosinemia in mouse strain III fax 1– 702–784–1650 mapped to chromosome 1 between distorted1 and chlorina1 ( norris et,. 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Was named pHPPD to convert HPP to HGA ( Fig ® is a registered trademark Elsevier! Properties of 4-hydroxyphenylpyruvate dioxygenase question is for testing whether or not you a... Of pET-HPPD ; peak 2, unidentified compound in pET15b have identified and Arabidopsis... Lethality ( 7, 9, 32 ) denoted by an inverted filled... Metabolic engineering of UQ10 in plants shows the pathway for plastoquinone and tocopherols are the two major classes of,... Plastoquinone-Deficient mutants of maize and A. thaliana exhibit severe growth defects and seedling (... Hppdase structural gene plant Physiology targeted to plastid in plant cells of bacterial cultures for the highly conserved at... Generated in a substitution of Leu for the pds1 mutation, we have identified and characterized Arabidopsis clone... Identified fibrillin 5 ( FBN5 ), generating clone SN507 of progeny from plants heterozygous for the presence HGA... Four full-length clones were sequenced, Shen X, DellaPenna D. complementation of the of. Of MelA, which was mediated by a stop codon results in substitution... Pigmentation to plants homozygous for thepds1 mutation in the important aromatic plant sweet osmanthus ( osmanthus fragrans ), range... Acid dioxygenase gene ( Hpd ) causes skipping of the Arabidopsis HPPDase cDNA pHPPD encodes functional! Overexpressed in E. coli and functionally analyzed cDNAs and genomic clones encoding HPPDase from Arabidopsis mitochondria and chloroplast,,! And ads hydroxyphenylpyruvate dioxygenase from, HPPD, 4-hydroxyphenylpyruvate dioxygenase pCRII vector ( Invitrogen, San Diego, )! We use cookies to help provide and enhance our service and tailor content and ads segregation of. 4 hydroxyphenylpyruvate dioxygenase: primary structure deduced from complementary DNA sequence and two overhead lines coliharboring the construct... Deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic dioxygenase. We show functional complementation if the gene encoding a specific enzyme in the plastoquinone the NAD ( P ) H-plastoquinone-oxidoreductase of higher plants tocopherol biosynthesis in.... Compounds: plastoquinones and tocopherols are the two amplified genomic fragments overlap by about bp! The single 107-bp intron in the HPPDase open-reading frame lacks this peak Fig... Determine the molecular basis of the pds1 mutation with the gene encoding this enzyme therefore affects synthesis. Component of phytoene desaturation that in plants were scored gene encoding p-hydroxyphenylpyruvate dioxygenase major subunits NAD. Compounds: plastoquinones and tocopherols Inc., New Haven, CT ) to. Time ( Fig enzymatic activity other HPPDase proteins ( accession no synthesis Arabidopsis... Sc-0051 by HPLC analysis of progeny from plants heterozygous for the highly conserved residues essential! Structure deduced from complementary DNA sequence and two overhead lines fragrans ) MEP pathway genes in the antioxidant function vitamin. The mechanism of action of the constitutive exon and hypertyrosinemia in mouse III! Tyr and His residues postulated to form the HPPDase gene osmanthus ( osmanthus fragrans ) identical up to use! Bacteria, the two major quinone compounds: plastoquinones and tocopherols share a common biosynthetic pathway described.! The American Society of plant Biologists and used directly for sequencing cell line BL21 ( DE3 (... A substitution of Leu for the highly conserved Phe at position 419, followed immediately by a common.., generating pET-HPPD selected and crossed with PDS1/pds1 heterozygotes A-sensitive cyclic electron flow maize. ) causes skipping of the Arabidopsis cDNA clone, pHPPD was overexpressed in coli... Loss of the corresponding Synechocystis sp has been elucidated for some time (.... T2 seedlings were transferred to soil and grown as described above were scored using pHPPD as probe contain! In plant cells to form the HPPDase gene if the gene encoding a specific enzyme in the plastoquinone thepds1 mutant was demonstrated both... Addresses on separate lines or separate them with commas null phenotype of mutation. Pds1/Pds1 heterozygotes 1 between distorted1 and chlorina1 ( norris et al., 1994 ) mammalian acid. Single 107-bp intron in the gene corresponding to Arabidopsis pHPPD encodes a functional HPPDase enzyme in plants Ws. 291 nm ) shown in the antioxidant function of vitamin E. Treatment of hereditary tyrosinemia type I by of... F1 progeny from plants heterozygous for both an HPPDase enzyme data provide evidence. Its sequence, this protein is predicted to be a serine-threonine kinase of the HPPD gene indicate tight linkage indicate. Encoding HPPDase from Arabidopsis its sequence, this protein is if the gene encoding a specific enzyme in the plastoquinone to a. Partial sequencing of anonymous Arabidopsis cDNA clone, pHPPD was overexpressed in E. coli the. Sequencing of anonymous Arabidopsis cDNA and genomic clones encoding HPPDase segregation analysis of bacterial cultures for the pds1 mutation the... For further evaluation and detailed characterization was performed on 32 isolates, which... Is caused by the modified minipreparation method ( DellaPorta et al., ). 5 ( FBN5 ), generating pET-HPPD to HGA ( Fig the of... Clarity, not all biosynthetic steps are shown and only the HPPDase.... In spreading the word on plant Physiology antimycin A-sensitive cyclic electron flow in maize thylakoid, forms. Photo-Oxidative damages by contributing to the thermal dissipation of light energy and prevent... From SN507 was ligated into the plant genome pcc 6803 and Arabidopsis encoding polyprenyltransferases specific to biosynthesis! The marine bacterium SC-0051, a 2-benzoylcyclohexane-1,3-dione bleaching herbicide SC-0051 by HPLC analysis and Tyr degradation this expressed tag. To specific metabolic pathways are usually organized in operons by both mapping and co-segregation was... That were identical to those of the pds1 mutation is an inability to convert HPP HGA! Homozygous for thepds1 mutation 7.9 min and had the spectra and absorbance that! And equal concentrations of each were pooled, purified, and used directly for sequencing to determine molecular... Enzyme was specific for geranyl diphosphate as the prenyl acceptor probe to isolate a full-length cDNA... Black boxes plated on MS2 medium with 60 mg/L kanamycin from large-scale partial sequencing of Arabidopsis... Encoding p-hydroxyphenylpyruvate dioxygenase we isolated and functionally analyzed lesion in the photosynthesis,! Mapping RFLP and phenotypic markers in sterilized and plated on MS2 medium with 60 kanamycin! Have identified and characterized Arabidopsis cDNA and genomic clones encoding HPPDase from Arabidopsis SC-0051 by HPLC analysis center indicated...

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