1). of chlorophyll biosynthesis enzymes and that of the Lhcb genes in the model organism Dunaliella salina. 1 ). 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. Kanamycin-resistant F1 seedlings were transferred to soil and grown as described above. Arabidopsis HPPDase amino acid residues showing identity in 9 of the other 13 HPPDase proteins are indicated with shaded boxes. SC-0051, a 2-benzoylcyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme. AF060481) and pds1 mutant tissues were determined by direct sequencing of PCR-amplified products. Sequence analysis of the wild-type and mutant HPPDase genomic sequences identified a small deletion that produces a truncated protein in the mutant. 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. Our results indicate that in … This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. 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. The conjugated rings of HPP and HGA are numbered to indicate rearrangement of the side chain. Subunit 2 of NADH-dehydrogenase is one of the major subunits in NAD dehydrogenase complex. The location of the single 107-bp intron in the HPPDase genomic sequences of Ws andpds1 is denoted by an inverted, filled triangle. Finally, we show functional complementation of the pds1 mutant phenotype when the HPPDase cDNA is constitutively expressed. The functional expression of the Arabidopsis HPPDase cDNA in E. coli demonstrates that it encodes a functional HPPDase enzyme. Confers resistance to photo-oxidative damages by contributing to the thermal dissipation of light energy and to lumenal acidification (increase of pH gradient). 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. Genes encoding the DOXP pathway enzymes are found in the nucleus, but their products are targeted to the chloroplast (Lange et al., 1998). Copyright © 2002 Federation of European Biochemical Societies. The protein sequence is shown in boldface underneath the nucleotide sequence (accession no. Nucleotide and deduced amino acid sequence of the Arabidopsis HPPDase cDNA pHPPD. 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. 1; Norris et al., 1995). Genomic DNA for co-segregation analysis was isolated from F2 progeny by the modified minipreparation method (DellaPorta et al., 1983). 2). T20952) with homology to the carboxy terminus of human HPPDase (accession no.X72389). The hydroxyphenylpyruvate dioxygenase from, HPPD, 4-hydroxyphenylpyruvate dioxygenase. Three independent transgenic lines constitutively overexpressing HPPDase were selected and crossed withPDS1/pds1 heterozygotes. 3A) and had a spectrum and absorbance maximum that were identical to those of the HGA standard (Fig. The pET-HPPD culture filtrate had a prominent peak that co-migrated with the HGA standard (Fig. Loss of the transgene should restore a 3:1 green:white ratio to such plants. 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). Peripheral neuropathy as the presenting feature of tyrosinaemia type I and effectively treated with an inhibitor of 4-hydroxyphenylpyruvate dioxygenase. Using desert plant Calotropis (Calotropis procera), this study focuses on the RNA editing … 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. Constitutive expression of the protein encoded by the Arabidopsis HPPDase cDNA is sufficient to restore wild-type pigmentation to plants homozygous for thepds1 mutation. To functionally test the hypothesis that the Arabidopsispds1 mutation is the result of a lesion in the structural HPPDase gene, it is necessary to isolate and functionally characterize Arabidopsis HPPDase cDNAs and the corresponding wild-type and mutant HPPDase alleles. (1995) showed a Fd-dependent, antimycin A-sensitive cyclic electron flow in maize thylakoid, which was mediated by a novel cytochrome b . In our recent study, the first cDNA encoding flavanone-specific plant prenyltransferase, naringenin 8-dimethylallyltransferase (SfN8DT-1) from Sophora flavescens, was reported 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). Plastoquinone-deficient mutants of maize and A. thaliana exhibit severe growth defects and seedling lethality (7, 9, 32). Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium. 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 ). Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase. DNA sequencing was performed using a dye deoxy terminator cycle sequencing kit (Applied Biosystems) and an automated DNA sequencer (model 310, Applied Biosystems). 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 … In this study, we isolated and identified 10 MEP pathway genes in the important aromatic plant sweet osmanthus (Osmanthus fragrans). The prenyl alcohol product clearly indicated that the cloned gene catalyzed the synthesis of a C 45 prenyl moiety . The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria differs substantially from that in plants. Together, these data indicate that the PDS1 locus and HPPDase gene are linked in the Arabidopsis genome. Similarly, for the pds1 mutant, two sets of primers were used: SN418T7+10 and SN418MF+1b (5′-CAGATGTTGTAGCCCT-3′) for the first 1000 bp of the gene, and SN418T7+4 and SN418MF+12 for the last 700 bp of the gene. Plastoquinones are fundamentally important components of the photosynthetic electron-transport chain, whereas tocopherols are thought to be important for free radical scavenging and protection from oxidative stress. This brown coloration is caused by the accumulation of ochronotic pigment, which forms upon the oxidative polymerization of HGA. These data demonstrate that the Arabidopsis cDNA pHPPD encodes a functional HPPDase enzyme. DOI: https://doi.org/10.1104/pp.117.4.1317. Digestion of Ws and Col genomic DNA withNcoI gave a restriction fragment-length polymorphism for the pHPPD probe. pET15b and pET-HPPD were transformed into Escherichia coli cell line BL21(DE3) (Novagen) via electroporation. Norris SR, Shen X, DellaPenna D. Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase. To determine the molecular basis of the pds1 mutation, the HPPDase genomic DNA sequences from wild-type Ws Arabidopsis (accession no. This result defines the molecular basis of the pds1 mutation as a mutation in the structural HPPD gene. Tatiana FEDORCHUK | Cited by 123 | of University of Toronto, Toronto (U of T) | Read 12 publications | Contact Tatiana FEDORCHUK This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene. B, Absorption spectra of peaks 1 and 2 from A. In conclusion, we have identified and characterized Arabidopsis cDNA and genomic clones encoding HPPDase. Linkage of the HPPD gene and thepds1 mutant was demonstrated by both mapping and co-segregation analysis. The PDS1 gene product could therefore be the HPPDase enzyme, a regulator of HPPDase expression or activity, or a cofactor required for HPPDase activity. For clarity, not all biosynthetic steps are shown and only the HPPDase reaction is shown in detail. The blots were hybridized with the pHPPD probe and washed two times at room temperature for 15 min with 2× SSC, 0.1% SDS and two times at 55°C for 25 min in 1× SSC, 0.1% SDS. 5). 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. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. They accumulate in chromoplasts and sequester carotenoids during the development of flowers and fruits. F2 progeny heterozygous for the pds1 mutation were selected from a cross betweenPDS1/pds1 (ecotype Ws) and PDS1/PDS1 (ecotype Columbia [Col]). The pds mutations thus provide genetic evidence that plastoquinone is an essential component for carotenoid biosynthesis in plants and provide insight into plastidic quinone synthesis and function. Copyright © 2021 Elsevier B.V. or its licensors or contributors. E. coli containing a control plasmid without the HPPDase open-reading frame lacks this peak (Fig. Clone SN500 was generated by subcloning a 1.5-kbKpnI/HindIII fragment containing the complete coding region of pHPPD into the plant-transformation shuttle vector pART7 (Gleave, 1992). Volume 45, issue 5 of the journal Zeitschrift für Naturforschung C was published in 1990. Figure 1 shows the pathway for plastoquinone and tocopherol biosynthesis in plants. In plants, triketones such as sulcotrione (2-[4-chloro-2-nitrobenzoyl]-5,5-dimethylcyclohexane-1,3-dione) are effective bleaching herbicides. To further understand the nature of the pds1 mutation, we have isolated and functionally analyzed cDNAs and genomic clones encoding HPPDase from Arabidopsis. 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). χ2 analysis shows that the ratio of green to white embryos in each line is statistically significant for a 15:1 ratio (Table I), indicating that the pds1 mutant phenotype was complemented by the presence of the overexpressed pHPPD cDNA in all plant lines analyzed. 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. 3B). 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. Kanamycin-resistant T2 seedlings were transferred to soil and grown to maturity, and T3 seed was harvested. This confirmed that the expressed enzyme is solanesyl diphosphate synthase. 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. 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. 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. SN506 was electroporated into Agrobacterium tumefaciens strain C58 and used to transform wild-type Arabidopsis (ecotype Ws) via vacuum infiltration (Bent et al., 1994). 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). The role of metabolism in the 4-hydroxyphenylpyruvic acid dioxygenase gene ( Hpd ) causes skipping of pds1. D. complementation of the HGA standard ( Fig encoding this enzyme therefore affects the synthesis both! Large amounts of two biosynthetically related quinone compounds in higher plants consequences of the... The expression of the carotenoid biosynthetic intermediate phytoene and photooxidation of the pds1 mutation with gene! 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