Peak 1, HGA standard and co-migrating peak in medium of pET-HPPD; peak 2, unidentified compound in pET15b. An important step in the early pathway is the formation of homogentisate (HGA) from 4‐hydroxyphenylpyruvate and molecular oxygen, catalyzed by the enzyme … 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. Our results indicate that in … RNA editing is common in terrestrial plants, especially in mitochondria and chloroplast. Plastoquinone and tocopherols share a common biosynthetic pathway that has been elucidated for some time (Fig. In both cases, the two amplified genomic fragments overlap by about 200 bp. Plastoquinone and tocopherols are the two major classes of chloroplastic, lipid-soluble quinone compounds in higher plants. Expression of Arabidopsis HPPDase cDNA inE. 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. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. PCR products were analyzed by gel electrophoresis, and equal concentrations of each were pooled, purified, and used directly for sequencing. To further understand the nature of the pds1 mutation, we have isolated and functionally analyzed cDNAs and genomic clones encoding HPPDase from Arabidopsis. These data demonstrate that the Arabidopsis cDNA pHPPD encodes a functional HPPDase enzyme. Genes encoding the DOXP pathway enzymes are found in the nucleus, but their products are targeted to the chloroplast (Lange et al., 1998). Moreover, the gene product was targeted to plastid in plant cells. The 17-bp deletion in the HPPDase gene in pds1 is denoted by a boldface, italic DNA sequence and two overhead lines. They accumulate in chromoplasts and sequester carotenoids during the development of flowers and fruits. 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). Plastoquinone-deficient mutants of maize and A. thaliana exhibit severe growth defects and seedling lethality (7, 9, 32). 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. 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. This disorder results from a deficiency in the last enzyme of Tyr catabolism (Lindstedt et al., 1992; Gibbs et al., 1993) and 2-(2-nitro-4-trifluromethylbenzoyl)-1,3-cyclohexanedione treatment inhibits liver HPPDase activity, blocking formation of HGA and its subsequent breakdown to the toxic intermediates succinylacetoacetate and succinylacetone. SC-0051, a 2-benzoylcyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme. Sequence analysis of the HPPDase gene from both wild-type and homozygous pds1 mutant plants was performed to define the molecular basis of the pds1 mutation. Inhibition of barnyardgrass 4-hydroxyphenylpyruvate dioxygenase by sulcotrione. E. coli harboring the pET-HPPD construct developed a dark-brown color, whereas cultures containing the empty pET15b vector did not (data not shown). 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. We do not capture any email address. 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). 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 genes were transferred to the nucleus from the cyanobacterial-related endosymbiont that later became the chloroplast (Sprenger et al., 1997), This confirmed that the expressed enzyme is solanesyl diphosphate synthase. 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. Three independent transgenic lines constitutively overexpressing HPPDase were selected and crossed withPDS1/pds1 heterozygotes. The hydroxyphenylpyruvate dioxygenase from, HPPD, 4-hydroxyphenylpyruvate dioxygenase. 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. 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. In higher plants, etioplast to chloroplast differentiation is characterized by dramatic ultrastructural changes of the plastid and a concomitant increase in chl The null phenotype of thepds1 mutant suggests that these 26 carboxy-terminal residues are essential for HPPDase enzymatic activity. 6/f complex, and to a lesser extent as an electron carrier for NAD(P)H-plastoquinone oxidoreductases (Berger et al., 1993). avermitilis HPPDase was expressed in E. coli(Denoya et al., 1994). The nucleotide sequence of the originally identified, truncated expressed sequence tag (accession no. 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. A, HPLC analysis of a HGA standard in Luria-Bertani broth is shown in the top plot. 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. 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). Transgenic plants overexpressing the pHPPD cDNA were generated in a wild-type background and crossed with plants heterozygous for the pds1 mutation. Figure 1 shows the pathway for plastoquinone and tocopherol biosynthesis in plants. This brown coloration is caused by the accumulation of ochronotic pigment, which forms upon the oxidative polymerization of HGA. T20952) is indicated by a single underline. coli. Linkage analysis indicated that the gene corresponding to Arabidopsis pHPPD maps near (±4 centimorgans) the pds1 mutation (data not shown). This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. 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). 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. Kanamycin-resistant F1 seedlings were transferred to soil and grown as described above. 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. 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. 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). In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. A similar dark-brown coloration was reported when the gene encoding HPPDase fromStreptomyces avermitilis was expressed in E. coli(Denoya et al., 1994). 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 resulting F1 seeds were surface sterilized and plated on MS2 medium with 60 mg/L kanamycin. Norris SR, Barrette TR, DellaPenna D. Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. 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. 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. 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. 1992); and recently it has been demonstrated that the enzyme participates in a ferredoxin-dependent Most significantly, these data define the molecular basis of the pds1 mutation as a lesion in the structural HPPD gene. This partial cDNA was used as a probe to isolate a full-length cDNA that was named pHPPD. Sequence analysis of the genome of the unicellular cyanobacterium. 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. In previous work we demonstrated that the biochemical basis of the Arabidopsis pds1 mutation is an inability to convert HPP to HGA (Fig. Co-segregation of the pds1 and HPPDase loci was determined by restriction fragment-length polymorphism linkage analysis using pHPPD as probe. 2). Loss of the transgene should restore a 3:1 green:white ratio to such plants. The first ATG of pHPPD begins an open-reading frame encoding a 50-kD protein of 445 amino acids (Fig. Both HPPDase genomic sequences contain a single 107-bp intron of identical sequence between positions 1162 and 1163 of the HPPD cDNA sequence in Figure 2. 3A). Future studies will determine the consequences of overexpressing the wild-type HPPDase enzyme in plants. 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. In our recent study, the first cDNA encoding flavanone-specific plant prenyltransferase, naringenin 8-dimethylallyltransferase (SfN8DT-1) from Sophora flavescens, was reported 1; Norris et al., 1995). 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. PCC 6803 and Arabidopsis encoding polyprenyltransferases specific to tocopherol biosynthesis. This stop codon results in the deletion of the remaining 26 amino acids from the carboxyterminal end of the protein. Norris SR, Shen X, DellaPenna D. Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase. The functional expression of the Arabidopsis HPPDase cDNA in E. coli demonstrates that it encodes a functional HPPDase enzyme. 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. 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). Studies on the expression of NDH-H, a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher plant chloroplasts. In plants, triketones such as sulcotrione (2-[4-chloro-2-nitrobenzoyl]-5,5-dimethylcyclohexane-1,3-dione) are effective bleaching herbicides. 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). E. coli containing a control plasmid without the HPPDase open-reading frame lacks this peak (Fig. These results demonstrate conclusively that the nature of thepds1 mutation in Arabidopsis is a mutation in the gene encoding the HPPDase enzyme. 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. Kanamycin-resistant T2 seedlings were transferred to soil and grown to maturity, and T3 seed was harvested. 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). From its sequence, this protein is predicted to be a serine-threonine kinase. 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 protein-sequence homology of the putative Arabidopsis HPPDase to other HPPDases suggested that it encodes an HPPDase enzyme. DOI: https://doi.org/10.1104/pp.117.4.1317. The CO2 lost and molecular oxygen introduced by HPPDase are indicated with a larger font and asterisks, respectively. 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. Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. 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). 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