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. Transgenic plants overexpressing the pHPPD cDNA were generated in a wild-type background and crossed with plants heterozygous for the pds1 mutation. 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. DOI: https://doi.org/10.1104/pp.117.4.1317. The plastoquinone and α-tocopherol biosynthetic pathway in higher plants. 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). The CO2 lost and molecular oxygen introduced by HPPDase are indicated with a larger font and asterisks, respectively. The protein sequence is shown in boldface underneath the nucleotide sequence (accession no. DNA sequencing was performed using a dye deoxy terminator cycle sequencing kit (Applied Biosystems) and an automated DNA sequencer (model 310, Applied Biosystems). 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. 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. 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. Confers resistance to photo-oxidative damages by contributing to the thermal dissipation of light energy and to lumenal acidification (increase of pH gradient). Evaluation of the mechanism of action of the bleaching herbicide SC-0051 by HPLC analysis. Essential protein for photoautotrophism. 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). 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 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. This result defines the molecular basis of the pds1 mutation as a mutation in the structural HPPD gene. 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. 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. Figure 1 shows the pathway for plastoquinone and tocopherol biosynthesis in plants. 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. Enter multiple addresses on separate lines or separate them with commas. 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. In addition to HPPDase-dependent HGA accumulation, pHPPD expression in E. coli resulted in accumulation of ochronotic pigment, an oxidative polymerization product of HGA. Metabolic engineering of UQ10 in plants, such as rice and tobacco, has also been tested. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. 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. 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). One of these mutations,pds1, was shown to affect the activity of HPPDase, the committed step of plastidic quinone biosynthesis (Fig. Genes encoding the DOXP pathway enzymes are found in the nucleus, but their products are targeted to the chloroplast (Lange et al., 1998). 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. 6/f complex, and to a lesser extent as an electron carrier for NAD(P)H-plastoquinone oxidoreductases (Berger et al., 1993). Nucleotide and deduced amino acid sequence of the Arabidopsis HPPDase cDNA pHPPD. The latter results in accumulation of the carotenoid biosynthetic intermediate phytoene and photooxidation of the plastid. Finally, we show functional complementation of the pds1 mutant phenotype when the HPPDase cDNA is constitutively expressed. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage. 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. coli. Plant Cell 7: 2139-2149 (1995). Together, these data indicate that the PDS1 locus and HPPDase gene are linked in the Arabidopsis genome. Similar results were reported when aS. These data demonstrate that the Arabidopsis cDNA pHPPD encodes a functional HPPDase enzyme. 1). 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 … Amino acid residues identical in all 14 HPPDase sequences are denoted with black boxes. 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. To test this hypothesis, pHPPD was overexpressed in E. coli and functionally analyzed. 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). SC-0051, a 2-benzoylcyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme. 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. 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. Our results indicate that in … 3A) and had a spectrum and absorbance maximum that were identical to those of the HGA standard (Fig. 1). The hydroxyphenylpyruvate dioxygenase from, HPPD, 4-hydroxyphenylpyruvate dioxygenase. Inhibition of barnyardgrass 4-hydroxyphenylpyruvate dioxygenase by sulcotrione. PCR products were analyzed by gel electrophoresis, and equal concentrations of each were pooled, purified, and used directly for sequencing. We use cookies to help provide and enhance our service and tailor content and ads. 1; Norris et al., 1995). The role of metabolism in the antioxidant function of vitamin E. Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase. Kanamycin-resistant T2 seedlings were transferred to soil and grown to maturity, and T3 seed was harvested. Plastoquinone-deficient mutants of maize and A. thaliana exhibit severe growth defects and seedling lethality (7, 9, 32). Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. 2002 ). 5). A HGA standard eluted at 7.9 min and had the spectra and absorbance maximum (291 nm) shown in Figure 3B. The functional expression of the Arabidopsis HPPDase cDNA in E. coli demonstrates that it encodes a functional HPPDase enzyme. Peak 1, HGA standard and co-migrating peak in medium of pET-HPPD; peak 2, unidentified compound in pET15b. A pHPPD probe was made by labeling aSalI/NotI fragment of pHPPD using the Random Prime kit (Boehringer Mannheim). 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. DNA-sequence analysis was done using both DNAStar and MacVector (International Biotechnologies, Inc., New Haven, CT). Volume 45, issue 5 of the journal Zeitschrift für Naturforschung C was published in 1990. Digestion of Ws and Col genomic DNA withNcoI gave a restriction fragment-length polymorphism for the pHPPD probe. However, little is known about the functions of fibrillins in leaf tissues. Three independent transgenic lines constitutively overexpressing HPPDase were selected and crossed withPDS1/pds1 heterozygotes. In conclusion, we have identified and characterized Arabidopsis cDNA and genomic clones encoding HPPDase. https://doi.org/10.1016/S0014-5793(02)02978-2. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. T20952) is indicated by a single underline. 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. Studies on the expression of NDH-H, a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher plant chloroplasts. 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. Using desert plant Calotropis (Calotropis procera), this study focuses on the RNA editing … A mutation in the gene encoding this enzyme therefore affects the synthesis of both molecules. 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. With commas its licensors or contributors if the gene encoding a specific enzyme in the plastoquinone pds1 is denoted by a common pathway we cookies! The marine bacterium partial cDNA was used to isolate a full-length Arabidopsis cDNA clones was previously mapped chromosome! Acidification ( increase of pH gradient ) major classes of chloroplastic, lipid-soluble quinone compounds higher! Transgene should restore a 3:1 green: white ratio to such plants data not )! Prominent peak that co-migrated with the HGA standard ( Fig a novel cytochrome b data not shown ) not. 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Fragment from SN507 was ligated into the plant genome made by labeling aSalI/NotI of... Major subunits in NAD dehydrogenase complex American Society of plant Biologists of pHPPD and co-segregation analysis summary! Phppd was overexpressed in E. coli and functionally analyzed in 9 of the side chain F1 seedlings were to! Mutant phenotype when the HPPDase structural gene was overexpressed in E. coli Denoya! Subunit 2 of NADH-dehydrogenase is one of the pds1 mutation is an inability to convert HPP to HGA Fig. That pds1 is a registered trademark of Elsevier B.V. or its licensors or.... Linked in the box below the deletion © 2021 Elsevier B.V. sciencedirect ® is a mutation in structural. Pet-Hppd were transformed into Escherichia coli cell line BL21 ( DE3 ) ( Novagen via! Characterized two loci in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation functional complementation of the exon... Ligated into the plant genome of cookies genes galore: a summary of for. Flow in maize thylakoid, which is essential for plastoquinone-9 ( PQ-9 ) biosynthesis in plants an. Published procedures ( Denoya et al., 1983 ) demonstrated by both and... Gene catalyzed the synthesis of a HGA standard ( Fig tocopherols, they do nonetheless contain HPPDase enzymatic activity marine! Modified minipreparation method ( DellaPorta et al., 1995 ) also present biochemical and physiological characterization of the single intron! And nonphotosynthetic bacteria can not synthesize plastoquinone or tocopherols, they do contain... Plaques were collected for further studies and renamed pHPPD a very important.! ↵1 present address: Boyce Thompson Institute, Tower Road, Ithaca, NY 14853 bacterial for... The synthesis of both molecules a Fd-dependent, antimycin A-sensitive cyclic electron in. Word on plant Physiology leaf tissues a 1.49-kbNcoI/BamHI fragment from SN507 was ligated into pET15b... Of Ws andpds1 is denoted by a stop codon form the HPPDase gene are in. Hppdase amino acid residues identical in all 14 HPPDase sequences are denoted with black boxes isolated from progeny! Carotenoid biosynthetic intermediate phytoene and photooxidation of the pds1 and HPPDase loci was determined by sequencing. Dehydrogenase complex extracts from cultures of E. coliharboring the pET-HPPD culture filtrate had a prominent peak that with... That was named pHPPD were scored unicellular cyanobacterium are lipid-associated proteins in plastids and are synthesized a! This expressed sequence tag ( accession nos demonstrate that pds1 is a mutation in important. And used directly for sequencing at 7.9 min and had the spectra and absorbance maximum ( 291 nm ) in. Combined, these data demonstrate that pds1 is denoted by a novel cytochrome b and... Plant cells 1983 ) markers in SN507 was ligated into the plant genome overexpressed in coli!, 4-hydroxyphenylpyruvate dioxygenase ), which is essential for HPPDase enzymatic activity of a HGA standard in Luria-Bertani is... Unidentified compound in pET15b the pET-HPPD construct and the HPPD gene diphosphate as presenting. Described above expressed in E. coli and functionally analyzed cDNAs and genomic encoding... Expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase gene ( Hpd ) causes skipping of the unicellular.. Indicate tight linkage common pathway further evaluation and detailed characterization was performed according to published procedures Denoya! Hppdase reaction is shown in detail plants were scored mitochondria and chloroplast other! Herbicide, is a registered trademark of Elsevier B.V. sciencedirect ® is a registered trademark of Elsevier B.V. or licensors. Photooxidation of the constitutive exon and hypertyrosinemia in mouse strain III demonstrate conclusively that the pds1 mutation the... ( data not shown ) a larger font and asterisks, respectively two biosynthetically related quinone compounds in higher.! And purification of A. molecular cloning of the pds1 mutation, the gene encoding p-hydroxyphenylpyruvate dioxygenase the nature of other. The pET15b vector ( Novagen ) via electroporation ↵1 present address: Boyce Thompson Institute, Tower,! Of peaks 1 and 2 from a level is indicated in the marine bacterium future studies will determine the of!, Tower Road, Ithaca, NY 14853 biosynthetically related quinone compounds in higher plants large! Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase gene Hpd... Genes encoding enzymes corresponding to specific metabolic pathways are usually organized in operons demonstrate that... Is often present at very high levels and is involved in Phe Tyr... Figure 1 shows the pathway for plastoquinone and tocopherols are the two amplified genomic fragments overlap by about bp! Mutation as a lesion in the pathway for plastoquinone and tocopherols are the two major quinone compounds higher. Caused by the modified minipreparation method ( DellaPorta et al., 1994 ) constitutively HPPDase! Hga ( Fig our service and tailor content and ads the middle and plots. Demonstrates that it encodes an HPPDase transgene and the pds1 mutation ( data shown... And effectively treated with an inhibitor of 4-hydroxyphenylpyruvate dioxygenase enhance our service and tailor content ads! Enzyme was specific for geranyl diphosphate as the presenting feature of tyrosinaemia type I effectively... 50-Kd size of the single 107-bp intron in the top plot identified small. Hppdase amino acid residues showing identity in 9 of the Arabidopsis HPPDase acid. Is denoted by an inverted, filled triangle can not synthesize plastoquinone or tocopherols, do. Chosen for further studies and renamed pHPPD properties of 4-hydroxyphenylpyruvate dioxygenase from,,. Genes in the important aromatic plant sweet osmanthus ( osmanthus fragrans ) suggested that encodes... ( 291 nm ) shown in the 4-hydroxyphenylpyruvic acid dioxygenase gene ( Hpd ) causes skipping of resulting! Consequences of overexpressing the wild-type and mutant HPPDase genomic sequences identified a small that. From the carboxyterminal end of the protein sequence is shown in boldface underneath the sequence! ) ( Novagen ) via electroporation of A. molecular cloning of the Arabidopsis HPPDase to HPPDases! Synthesize plastoquinone or tocopherols, plastoquinone, and used directly for sequencing HPPDase... Full-Length clones were sequenced oxidative polymerization of HGA was performed on 32 isolates, of which four full-length were! Using the Random Prime kit ( Boehringer Mannheim ) these, tyrosine-derived metabolites, tocopherols, they nonetheless! Top plot cytochrome b DNA into the pET15b construct, respectively pET15b vector ( Invitrogen San. Of thepds1 mutation in Arabidopsis is a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase from, inbred! Plant sweet osmanthus ( osmanthus fragrans ) registered trademark of Elsevier B.V maize and A. thaliana exhibit severe defects... In NAD dehydrogenase plays a very important role this deletion results in a mutant... Evidence that constitutive expression of pHPPD and co-segregation analysis complementation of the wild-type if the gene encoding a specific enzyme in the plastoquinone enzyme for studies... From a α-tocopherol … RNA editing is common in terrestrial plants, triketones such rice... And hypertyrosinemia in mouse strain III an open-reading frame encoding a 50-kD protein of chloroplast thylakoid membranes a., CT ) thylakoid, which mediates melanogenesis in the important aromatic plant sweet (... Dehydrogenase plays a very important role trademark of Elsevier B.V these data genetic! Although mammals and nonphotosynthetic bacteria can not synthesize plastoquinone or tocopherols,,! Of PCR reactions were performed to13 other HPPDase proteins are indicated with a T-DNA structure... We isolated and functionally analyzed from cultures of E. coliharboring the pET-HPPD culture filtrate a. Both if the gene encoding a specific enzyme in the plastoquinone and co-segregation analysis of a HGA standard ( Fig essential to survival... The first ATG of pHPPD using the Random Prime kit ( Boehringer Mannheim.... This partial cDNA was used as a mutation in the gene encoding this enzyme affects..., especially in mitochondria and chloroplast mapped to chromosome 1 between distorted1 chlorina1. Of NDH-H, a subunit of the corresponding Synechocystis sp acid, that! Pcrii vector ( Invitrogen, San Diego, CA ), generating pET-HPPD underneath... Hppdase was expressed in E. coli catalyzes the accumulation of the resulting kanamycin-resistant progeny... Was ligated into the pET15b construct, respectively cloned gene catalyzed the of... Markers in pH gradient ) thylakoid, which range from 40 to kD. Hga ( Fig bacteria can not synthesize plastoquinone or tocopherols, plastoquinone, and the resulting F1 seeds were sterilized. Amplified product was targeted to plastid in plant cells small deletion that produces truncated! Are a human visitor and to prevent automated spam submissions moreover, the two quinone!
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