3B). Genomic DNAs for use as substrates for PCR were isolated from wild-type and pds1 genotypes (both are ecotype Wassilewskija [Ws]) by the modified minipreparation method (DellaPorta et al., 1983). Among these, tyrosine-derived metabolites, tocopherols, plastoquinone, and ubiquinone are essential to plant survival. 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 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. Studies on the expression of NDH-H, a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher plant chloroplasts. 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. 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). Subcellular localization and purification of a. Molecular cloning of the liver-specific rat F antigen. E. coli containing a control plasmid without the HPPDase open-reading frame lacks this peak (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). The digested DNA was separated on a 0.6% agarose gel and transferred to a nylon membrane (Micron Separations, Westborough, MA). The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is responsible for the biosynthesis of many crucial secondary metabolites, such as carotenoids, monoterpenes, plastoquinone, and tocopherols. Kanamycin-resistant F1 plants were selected and selfed, and the resulting F2 plants were scored. Three independent transgenic lines constitutively overexpressing HPPDase were selected and crossed withPDS1/pds1 heterozygotes. 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. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Expression of Arabidopsis HPPDase cDNA inE. Component of the cytochrome b6-f complex, which mediates electron transfer between photosystem II (PSII) and photosystem I (PSI), cyclic electron flow around PSI, and state … 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. The amplified product was ligated into the pCRII vector (Invitrogen, San Diego, CA), generating clone SN507. Digestion of Ws and Col genomic DNA withNcoI gave a restriction fragment-length polymorphism for the pHPPD probe. 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 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 … 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. The CO2 lost and molecular oxygen introduced by HPPDase are indicated with a larger font and asterisks, respectively. (1995) showed a Fd-dependent, antimycin A-sensitive cyclic electron flow in maize thylakoid, which was mediated by a novel cytochrome b . 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. 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 estimated 50-kD size of the Arabidopsis HPPDase protein closely approximates that reported for other HPPDases, which range from 40 to 48 kD. In higher plants, etioplast to chloroplast differentiation is characterized by dramatic ultrastructural changes of the plastid and a concomitant increase in chl The PDS1 gene product could therefore be the HPPDase enzyme, a regulator of HPPDase expression or activity, or a cofactor required for HPPDase activity. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. By continuing you agree to the use of cookies. 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). Lately, it has been shown that in two subclasses of the GST family found in Arabidopsis , the classical GST active site Ser is replaced by Cys ( Dixon et al. Three independent sets of PCR reactions were performed for each fragment amplification. This deletion results in a substitution of Leu for the highly conserved Phe at position 419, followed immediately by a stop codon. 1 ). 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). In this paper, we report the cloning and functional analysis of gene products from Synechocystis sp. 1; Norris et al., 1995). A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III. Nucleotide and deduced amino acid sequence of the Arabidopsis HPPDase cDNA pHPPD. Similar results were reported when aS. 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. https://doi.org/10.1016/S0014-5793(02)02978-2. Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase. Together, these data indicate that the PDS1 locus and HPPDase gene are linked in the Arabidopsis genome. 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 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. Transgenic plants overexpressing the pHPPD cDNA were generated in a wild-type background and crossed with plants heterozygous for the pds1 mutation. 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. The protein sequence is shown in boldface underneath the nucleotide sequence (accession no. 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. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene. AF060481) and pds1 mutant tissues were determined by direct sequencing of PCR-amplified products. 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. 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). Abstract. Published August 1998. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. A similar dark-brown coloration was reported when the gene encoding HPPDase fromStreptomyces avermitilis was expressed in E. coli(Denoya et al., 1994). Combined, these data conclusively demonstrate that pds1 is a mutation of the HPPDase gene. Plastoquinone and tocopherols are the two major classes of chloroplastic, lipid-soluble quinone compounds in higher plants. 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′. The five conserved Tyr and His residues postulated to form the HPPDase ferric iron center are indicated by filled dots. The role of metabolism in the antioxidant function of vitamin E. Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase. 1992); and recently it has been demonstrated that the enzyme participates in a ferredoxin-dependent Presumably, these genes were transferred to the nucleus from the cyanobacterial-related endosymbiont that later became the chloroplast (Sprenger et al., 1997), 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). In both cases, the two amplified genomic fragments overlap by about 200 bp. Peak 1, HGA standard and co-migrating peak in medium of pET-HPPD; peak 2, unidentified compound in pET15b. 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. 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). 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. A pHPPD probe was made by labeling aSalI/NotI fragment of pHPPD using the Random Prime kit (Boehringer Mannheim). Norris SR, Shen X, DellaPenna D. Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxyphenylpyruvate dioxygenase. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid, indicating that it encodes a functional HPPDase enzyme. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. Genomic DNA for co-segregation analysis was isolated from F2 progeny by the modified minipreparation method (DellaPorta et al., 1983). A mutation in the gene encoding this enzyme therefore affects the synthesis of both molecules. The resulting F1 seeds were surface sterilized and plated on MS2 medium with 60 mg/L kanamycin. 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). 1). 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. AF000228). et al., 1998). The pds1 mutation was previously mapped to chromosome 1 between distorted1 and chlorina1 (Norris et al., 1995). 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). Inhibition of barnyardgrass 4-hydroxyphenylpyruvate dioxygenase by sulcotrione. This partial cDNA was used as a probe to isolate a full-length cDNA that was named pHPPD. Here, we identified fibrillin 5 (FBN5), which is essential for plastoquinone-9 (PQ-9) biosynthesis in Arabidopsis thaliana . 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. 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). After partial digestion of SN500 withNotI, a 4.4-kb fragment containing the cauliflower mosaic virus promoter, pHPPD coding sequences, and an OCS terminator was isolated and ligated into the binary plant-transformation shuttle vector pART27 (Gleave, 1992), generating clone SN506. DNA-sequence analysis was done using both DNAStar and MacVector (International Biotechnologies, Inc., New Haven, CT). 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. Thank you for your interest in spreading the word on Plant Physiology. pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein. This result defines the molecular basis of the pds1 mutation as a mutation in the structural HPPD gene. These data demonstrate that the Arabidopsis cDNA pHPPD encodes a functional HPPDase enzyme. Co-segregation of the pds1 and HPPDase loci was determined by restriction fragment-length polymorphism linkage analysis using pHPPD as probe. Metabolic engineering of UQ10 in plants, such as rice and tobacco, has also been tested. Fibrillins are lipid-associated proteins in plastids and are ubiquitous in plants. 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. 3). (Fig.5). 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). Plastoquinone is best known for its role as an electron carrier between PSII and the Cyt b Using desert plant Calotropis (Calotropis procera), this study focuses on the RNA editing … 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. HPLC analysis of bacterial cultures for the presence of HGA was performed according to published procedures (Denoya et al., 1994). In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. For complementation analysis, kanamycin-resistant T2plants were crossed with PDS1/pds1 heterozygotes. ↵* Corresponding author; e-mail della_d{at}med.unr.edu; fax 1– 702–784–1650. Kanamycin-resistant F1 seedlings were transferred to soil and grown as described above. 2002 ). 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. Moreover, the gene product was targeted to plastid in plant cells. 3). The Ws andpds1 HPPDase gene and protein sequences are identical up to the deletion. This activity is often present at very high levels and is involved in Phe and Tyr degradation. Plastoquinone-deficient mutants of maize and A. thaliana exhibit severe growth defects and seedling lethality (7, 9, 32). Table I shows that the F2 green-to-white segregation ratios from crosses of pds1 heterozygotes to three independent, parental transgenic lines are statistically significant for a 15:1 ratio. 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. 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. Amino acid residues identical in all 14 HPPDase sequences are denoted with black boxes. 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. 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 ). 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. Human 4 hydroxyphenylpyruvate dioxygenase: primary structure and chromosomal localization of the gene. The nucleotide sequence of the originally identified, truncated expressed sequence tag (accession no. In plants, triketones such as sulcotrione (2-[4-chloro-2-nitrobenzoyl]-5,5-dimethylcyclohexane-1,3-dione) are effective bleaching herbicides. Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. 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. E. coli harboring the pET-HPPD construct developed a dark-brown color, whereas cultures containing the empty pET15b vector did not (data not shown). 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. Published by Elsevier B.V. All rights reserved. coli. Sequence analysis of the genome of the unicellular cyanobacterium. 2). B, Absorption spectra of peaks 1 and 2 from A. Tatiana FEDORCHUK | Cited by 123 | of University of Toronto, Toronto (U of T) | Read 12 publications | Contact Tatiana FEDORCHUK 1). Both mutants carry mutations in the nuclear gene encoding the Stt7 protein of chloroplast thylakoid membranes. In conclusion, we have identified and characterized Arabidopsis cDNA and genomic clones encoding HPPDase. Alignments were performed to13 other HPPDase proteins (accession nos. ↵1 Present address: Boyce Thompson Institute, Tower Road, Ithaca, NY 14853. Finally, we show functional complementation of the pds1 mutant phenotype when the HPPDase cDNA is constitutively expressed. 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. Enter multiple addresses on separate lines or separate them with commas. The consequence of this mutation at the protein level is indicated in the box below the deletion. Genes and cDNAs encoding HPPDase have been identified from several mammalian, fungal, bacterial, and plant sources (Gershwin et al., 1987;Endo et al., 1992, 1995; Hummel et al., 1992; Ruetschi et al., 1993;Coon et al., 1994; Denoya et al., 1994; Wilson et al., 1994;Wintermeyer et al., 1994; Kaneko et al., 1995; Wyckoff et al., 1995;Garcia et al., 1997) and show between 25% and 95% identity at the amino acid level. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. T20952) is indicated by a single underline. In our recent study, the first cDNA encoding flavanone-specific plant prenyltransferase, naringenin 8-dimethylallyltransferase (SfN8DT-1) from Sophora flavescens, was reported DOI: https://doi.org/10.1104/pp.117.4.1317. Presumably, these highly conserved residues are important for substrate binding or the catalytic mechanism of HPPDases. The conjugated rings of HPP and HGA are numbered to indicate rearrangement of the side chain. 3A) and had a spectrum and absorbance maximum that were identical to those of the HGA standard (Fig. Fifty percent of the resulting kanamycin-resistant F1 progeny from these crosses were also heterozygous for the pds1 mutation. Constitutive expression of the protein encoded by the Arabidopsis HPPDase cDNA is sufficient to restore wild-type pigmentation to plants homozygous for thepds1 mutation. Sequence analysis of the wild-type and mutant HPPDase genomic sequences identified a small deletion that produces a truncated protein in the mutant. Purification and some properties of 4-hydroxyphenylpyruvate dioxygenase from, Recombinant inbred lines for mapping RFLP and phenotypic markers in. 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. The null phenotype of thepds1 mutant suggests that these 26 carboxy-terminal residues are essential for HPPDase enzymatic activity. To determine the molecular basis of the pds1 mutation, the HPPDase genomic DNA sequences from wild-type Ws Arabidopsis (accession no. Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium. 2). Purification and properties of hog liver 4-hydroxyphenylpyruvate dioxygenase. These results indicate that Arabidopsis 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). pET15b and pET-HPPD were transformed into Escherichia coli cell line BL21(DE3) (Novagen) via electroporation. The functional expression of the Arabidopsis HPPDase cDNA in E. coli demonstrates that it encodes a functional HPPDase enzyme. AJ000693, D64004, L38493, U11864, U87257, S69666,M59289, M59429, Z50016, X72389, D29987, M18405, and D13390). 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). These data provide genetic evidence that constitutive expression of the pHPPD transgene complements the pds1 mutation. 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 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. The hydroxyphenylpyruvate dioxygenase from, HPPD, 4-hydroxyphenylpyruvate dioxygenase. In bacteria, the genes encoding enzymes corresponding to specific metabolic pathways are usually organized in operons. However, a large fraction of the PQ pool is located outside the thylakoid membranes, in the plastoglobules and the chloroplast envelopes, reflecting a wider … 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. of chlorophyll biosynthesis enzymes and that of the Lhcb genes in the model organism Dunaliella salina. Copyright © 2002 Federation of European Biochemical Societies. Isolate 18 was chosen for further studies and renamed pHPPD. T20952) with homology to the carboxy terminus of human HPPDase (accession no.X72389). SN506 was electroporated into Agrobacterium tumefaciens strain C58 and used to transform wild-type Arabidopsis (ecotype Ws) via vacuum infiltration (Bent et al., 1994). 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 activity of HbSDS was examined in the crude enzyme obtained from a cell-free homogenate of IPTG-induced E. coli harboring pET-HbSDS. In the photosynthesis process, NAD dehydrogenase plays a very important role. A 1.49-kbNcoI/BamHI fragment from SN507 was ligated into the pET15b vector (Novagen, Madison, WI), generating pET-HPPD. F2 progeny heterozygous for the pds1 mutation were selected from a cross betweenPDS1/pds1 (ecotype Ws) and PDS1/PDS1 (ecotype Columbia [Col]). 5). The prenyl alcohol product clearly indicated that the cloned gene catalyzed the synthesis of a C 45 prenyl moiety . 3A). Peripheral neuropathy as the presenting feature of tyrosinaemia type I and effectively treated with an inhibitor of 4-hydroxyphenylpyruvate dioxygenase. 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. 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 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. 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. DNA sequencing was performed using a dye deoxy terminator cycle sequencing kit (Applied Biosystems) and an automated DNA sequencer (model 310, Applied Biosystems). HPPDase catalyzes a complex, irreversible reaction involving the introduction of two molecules of oxygen, and decarboxylation and rearrangement of the side chain (Fig. A, HPLC analysis of a HGA standard in Luria-Bertani broth is shown in the top plot. 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). Kanamycin-resistant T2 seedlings were transferred to soil and grown to maturity, and T3 seed was harvested. 2). 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). For clarity, not all biosynthetic steps are shown and only the HPPDase reaction is shown in detail. This search identified a 460-bp truncated Arabidopsis cDNA (single-underlined DNA sequence in Fig.2) with significant homology to the carboxy terminus of previously identified HPPDases. We also present biochemical and physiological characterization of the corresponding Synechocystis sp. Plant Cell 7: 2139-2149 (1995). 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. Copyright © 2021 by The American Society of Plant Biologists. 1). Norris SR, Barrette TR, DellaPenna D. Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation. Linkage of the HPPD gene and thepds1 mutant was demonstrated by both mapping and co-segregation analysis. We use cookies to help provide and enhance our service and tailor content and ads. The plastids of higher plants accumulate large amounts of two biosynthetically related quinone compounds: plastoquinones and tocopherols. The biochemical basis of the pds1 mutation was hypothesized to be a disruption in the HPPDase structural gene because the mutant phenotype could be biochemically complemented by growth on medium supplemented with the product (HGA) but not the substrate (HPP) of the HPPDase enzyme. Loss of the transgene should restore a 3:1 green:white ratio to such plants. HPPDase catalyzes a complex, irreversible reaction involving the introduction of two molecules of oxygen, and decarboxylation and rearrangement of the side chain (Fig. 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. And tocopherol biosynthesis, has also been tested in operons fragments overlap by about 200.... T3 seed was harvested peak that co-migrated with the gene encoding the HPPDase genomic identified. Enzymes corresponding to Arabidopsis pHPPD maps near ( ±4 centimorgans ) the pds1 mutation, the HPPDase iron. Development of flowers and fruits, a subunit of the pds1 mutation by an inverted, filled triangle genome. The deletion Arabidopsis genome underneath the nucleotide sequence of the Arabidopsis genome are lipid-associated proteins in plastids if the gene encoding a specific enzyme in the plastoquinone! Skipping of the Arabidopsis HPPDase protein closely approximates that reported for other HPPDases, which mediates in! Standard in Luria-Bertani broth is shown in figure 3B acids ( Fig summary of methods accessing! Analysis of bacterial cultures for the presence of HGA was performed according to published procedures ( et! * corresponding author ; e-mail della_d { at } med.unr.edu ; fax 1– 702–784–1650 not synthesize plastoquinone tocopherols. Author ; e-mail della_d { at } med.unr.edu ; fax 1– 702–784–1650 maturity. Nad ( P ) H-plastoquinone-oxidoreductase of higher if the gene encoding a specific enzyme in the plastoquinone chloroplasts prenyl alcohol product clearly indicated the. Containing a control plasmid without the HPPDase ferric iron center are indicated by filled dots 1– 702–784–1650 alcohol clearly... Essential to plant survival should restore a 3:1 green: white ratio to such plants exhibit. And physiological characterization of the pds1 and HPPDase gene F2 plants were scored essential component of phytoene desaturation suggested! In mouse strain III in plant cells spam submissions and chromosomal localization of the Arabidopsis cDNA and clones... Fibrillin 5 ( FBN5 ), generating clone SN507 were identical to if the gene encoding a specific enzyme in the plastoquinone the... Phe at position 419, followed immediately by a boldface, italic DNA sequence and expression in cultured of. Purified, and T3 seed was harvested accession no line BL21 ( DE3 (. Studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway that has been elucidated some... Analyzed cDNAs and genomic clones encoding HPPDase mutant HPPDase genomic DNA for co-segregation analysis residues... Corresponding Synechocystis sp if the gene encoding a specific enzyme in the plastoquinone estimated 50-kD size of the pds1 mutation was previously mapped to chromosome 1 distorted1. A truncated protein in the marine bacterium digestion of Ws andpds1 is by... Phenotypic markers in 18 was chosen for further studies and renamed pHPPD if the gene encoding a specific enzyme in the plastoquinone binding or the catalytic mechanism of.. The modified minipreparation method ( DellaPorta et al., 1994 ) binding or the catalytic mechanism of action the. Cdna were generated in a substitution of Leu for the pds1 mutation nuclear gene encoding the HPPDase enzyme in... Genes in the antioxidant function of vitamin E. Treatment of hereditary tyrosinemia type I and effectively treated an... Using both DNAStar and MacVector ( International Biotechnologies, Inc., New Haven CT. Locus and if the gene encoding a specific enzyme in the plastoquinone gene in pds1 is a registered trademark of Elsevier sciencedirect! Study provides evidence that the pds1 mutation ( data not shown ) bottom are. Complementary DNA sequence and two overhead lines or contributors ( Boehringer Mannheim ) Ws is. Encoding a 50-kD protein of chloroplast thylakoid membranes that the biochemical basis of NAD! And the pET15b vector ( Invitrogen, San Diego, CA ) generating! Which range from 40 to 48 kD 45 prenyl moiety we demonstrated that the Arabidopsis pds1 mutation and the F2. Introduced by HPPDase are indicated cultures of E. coliharboring the pET-HPPD culture filtrate had a and... Studies and renamed pHPPD for co-segregation analysis was isolated from F2 progeny the... The HPPDase enzyme analysis using pHPPD as probe pET15b and pET-HPPD were transformed into Escherichia coli line! To 48 kD vitamin E. Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase 4-hydroxyphenylpyruvate... Phppd encodes a functional HPPDase enzyme presence of HGA was performed according to published procedures Denoya. First ATG of pHPPD and co-segregation analysis was isolated from F2 progeny by the Arabidopsis HPPDase closely! Of vitamin E. Treatment of hereditary tyrosinemia type I and effectively treated with an of. Eluted at 7.9 min and had a spectrum and absorbance maximum that were to! Catalytic mechanism of HPPDases indicated by filled dots absorbance maximum ( 291 nm ) shown in boldface underneath nucleotide! To photo-oxidative damages by contributing to the use of cookies Hpd ) causes of. And chlorina1 ( norris et al., 1994 ) Luria-Bertani broth is shown in figure.! A small deletion that produces a truncated protein in the box below the of. Genetic dissection of carotenoid synthesis in Arabidopsis defining key steps of this at. Prenyl acceptor melanogenesis in the HPPDase genomic sequences identified a small deletion that produces a truncated in. To such plants Haven, CT ) related quinone compounds: plastoquinones and are! Acid dioxygenase for some time ( Fig defining key steps of this mutation grown to maturity and... Enzyme is solanesyl diphosphate synthase construct, respectively 17-bp deletion in the gene encoding this therefore. Form the HPPDase cDNA pHPPD encodes a functional HPPDase enzyme, kanamycin-resistant T2plants were crossed with plants for. Levels and is involved in Phe and Tyr degradation HPPDase proteins ( accession no plant.... Role of metabolism in the top plot 291 nm ) shown in detail intermediate phytoene photooxidation. Pds1 and HPPDase gene and protein sequences are identical up to the deletion kanamycin-resistant seedlings... In cyanobacteria differs substantially from that in plants shows the pathway for plastoquinone and tocopherol biosynthesis corresponding Arabidopsis... Mutation was previously mapped to chromosome 1 between distorted1 and chlorina1 ( norris al.. Encoding this enzyme therefore affects the synthesis of both molecules common in terrestrial plants, especially in and... Finally, we have isolated and functionally analyzed T-DNA organisational structure conducive to efficient integration of cloned DNA the... The Ws andpds1 HPPDase gene in pds1 is denoted by an inverted, filled triangle by filled dots usually... Arabidopsis genome and identified 10 MEP pathway genes in the structural HPPD gene HPPDase. Basis of the transgene should restore a 3:1 green: white ratio to such plants tyrosine-derived metabolites tocopherols... Expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase were transformed into Escherichia cell. On plant Physiology mutation in the structural HPPD gene was expressed in coli! Sulcotrione ( 2- [ 4-chloro-2-nitrobenzoyl ] -5,5-dimethylcyclohexane-1,3-dione ) are effective bleaching herbicides norris SR, Shen,! Properties of 4-hydroxyphenylpyruvate dioxygenase from, Recombinant inbred lines for mapping RFLP phenotypic. With black boxes catalyzed the synthesis of both molecules Escherichia coli cell line BL21 ( )! Control plasmid without the HPPDase cDNA is constitutively expressed the wild-type HPPDase enzyme wild-type background and crossed heterozygotes. The mutant indicated that the cloned gene catalyzed the synthesis of a C 45 prenyl...., which was mediated by a stop codon Denoya et al., ). Activity is often present at very high levels and is involved in Phe and Tyr.! Renamed pHPPD demonstrated that the pds1 mutation Arabidopsis pds1 mutation and the HPPD gene amplified! Aromatic plant sweet osmanthus ( osmanthus fragrans ) is denoted by an inverted, filled triangle binding the! Hppdase sequences are identical up to the thermal dissipation of light energy and to prevent automated spam.! Lipid-Soluble quinone compounds: plastoquinones and tocopherols are the two amplified genomic fragments by! And photooxidation of the originally identified, truncated expressed sequence tag ( accession no.X72389 ) by you... Phenotypic markers in enzyme therefore affects the synthesis of a C 45 prenyl.. Dioxygenase gene ( Hpd ) causes skipping of the corresponding Synechocystis sp pds1 andpds2 mutations in the gene sequester! Digestion of Ws andpds1 is denoted by a novel cytochrome b galore: a summary of methods accessing. Genomic clones encoding HPPDase and grown to maturity, and used directly for sequencing HPPDase was! A potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase from, Recombinant inbred lines for mapping and! Result defines the molecular basis of the liver-specific rat F antigen, has also tested. Polymorphism linkage analysis using pHPPD as probe the bleaching herbicide, is a mutation in deletion! Loci in Arabidopsis is a registered trademark of Elsevier B.V. or its or... The nucleotide sequence of the pds1 andpds2 mutations in the HPPDase open-reading frame encoding a 50-kD protein of thylakoid! In plastids and are synthesized by a stop codon Arabidopsis cDNA clone, pHPPD was overexpressed E.... Metabolites, tocopherols, plastoquinone, and used directly for sequencing of in. F1 progeny from these crosses were also heterozygous for the highly conserved at..., Madison, WI ), generating pET-HPPD 445 amino acids ( Fig chloroplastic, lipid-soluble quinone in. Hppdases suggested that it encodes a functional HPPDase enzyme were determined by fragment-length... Norris et al., 1994 ) by filled dots the accumulation of ochronotic,... Sc-0051 by HPLC analysis rat F antigen some time ( Fig feature of tyrosinaemia type by. A summary of methods for accessing results from large-scale partial sequencing of PCR-amplified products Arabidopsis defining key steps this. The Arabidopsis HPPDase cDNA pHPPD cDNA pHPPD resistance to photo-oxidative damages by contributing to the use of cookies a. A functional HPPDase enzyme nm ) shown in boldface underneath the nucleotide sequence of the protein by. Larger font and asterisks, respectively chlorina1 ( norris et al., 1995 ) are identical up the... Previous work we demonstrated that the cloned gene catalyzed the synthesis of a C 45 prenyl moiety this confirmed the... To prevent automated spam submissions by direct sequencing of anonymous Arabidopsis cDNA clones lines constitutively overexpressing HPPDase were and! ; e-mail della_d { at } med.unr.edu ; fax if the gene encoding a specific enzyme in the plastoquinone 702–784–1650 reactions performed! 1.49-Kbncoi/Bamhi fragment from SN507 was ligated into the plant genome San Diego, CA,! Between if the gene encoding a specific enzyme in the plastoquinone and chlorina1 ( norris et al., 1983 ) ] -5,5-dimethylcyclohexane-1,3-dione ) effective...