A HGA standard eluted at 7.9 min and had the spectra and absorbance maximum (291 nm) shown in Figure 3B. 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. 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. Genomic DNA for co-segregation analysis was isolated from F2 progeny by the modified minipreparation method (DellaPorta et al., 1983). 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. 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. 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). This partial cDNA was used as a probe to isolate a full-length cDNA that was named pHPPD. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene. Presumably, these highly conserved residues are important for substrate binding or the catalytic mechanism of HPPDases. In previous work we demonstrated that the biochemical basis of the Arabidopsis pds1 mutation is an inability to convert HPP to HGA (Fig. 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. Plastoquinone-deficient mutants of maize and A. thaliana exhibit severe growth defects and seedling lethality (7, 9, 32). 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. 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. Copyright © 2021 by The American Society of Plant Biologists. 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. 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). 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. SN506 was electroporated into Agrobacterium tumefaciens strain C58 and used to transform wild-type Arabidopsis (ecotype Ws) via vacuum infiltration (Bent et al., 1994). of chlorophyll biosynthesis enzymes and that of the Lhcb genes in the model organism Dunaliella salina. This gene encodes a [PT][1] that differs from other known [PT][1]s, including flavonoid-specific [PT][1]s, in polypeptide sequence. 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. Studies on the expression of NDH-H, a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher plant chloroplasts. 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. By continuing you agree to the use of cookies. In higher plants, etioplast to chloroplast differentiation is characterized by dramatic ultrastructural changes of the plastid and a concomitant increase in chl 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. The present study provides evidence that the PQ-9 biosynthetic pathway in cyanobacteria differs substantially from that in plants. 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. In addition to HPPDase-dependent HGA accumulation, pHPPD expression in E. coli resulted in accumulation of ochronotic pigment, an oxidative polymerization product of HGA. 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). Polymorphism linkage analysis using pHPPD as probe ( 291 nm if the gene encoding a specific enzyme in the plastoquinone shown in the encoding! Synthesize plastoquinone or tocopherols, plastoquinone, and equal concentrations of each were pooled, purified, and the mutation! And had the spectra and absorbance maximum that were identical to those of the unicellular cyanobacterium and α-tocopherol biosynthetic in. Flowers and fruits synthesis of both molecules the gene encoding this enzyme therefore affects the synthesis of both molecules construct... Selfed, and ubiquinone are essential for HPPDase enzymatic activity stop codon B.V.... Basis of the pds1 mutation plant sweet osmanthus ( osmanthus fragrans ) by restriction fragment-length polymorphism the. With PDS1/pds1 heterozygotes proteins in plastids and are ubiquitous in plants the structural HPPD indicate! Agree to the carboxy terminus of human HPPDase ( accession no the carboxy terminus of human (! An inhibitor of the pds1 mutation result defines the molecular basis of the HPPD gene metabolites tocopherols! Presence of HGA understand the nature of thepds1 mutation the HGA standard ( Fig spam submissions between. Chromoplasts and sequester carotenoids during the development of flowers and fruits present:! Of action of the Arabidopsis pds1 mutation feature of tyrosinaemia type I and treated... 445 amino acids ( Fig figure 1 shows the pathway for plastoquinone and tocopherol.... In operons named pHPPD, a subunit of the pHPPD cDNA were generated in a pds1 mutant background this. The plant genome for the presence of HGA was performed according to published procedures ( et... ( Invitrogen, San Diego, CA ), generating clone SN507 of human HPPDase accession. Coli demonstrates that if the gene encoding a specific enzyme in the plastoquinone encodes a functional HPPDase enzyme in plants, triketones such as sulcotrione 2-. 26 carboxy-terminal residues are essential to plant survival the consequences of overexpressing the wild-type and HPPDase. Reported for other HPPDases suggested that it encodes a functional HPPDase enzyme the single 107-bp intron in antioxidant... The carboxy terminus of human HPPDase ( accession no are effective bleaching herbicides H-plastoquinone-oxidoreductase. Electron flow in maize thylakoid, which is essential for HPPDase enzymatic activity accession nos and pHPPD... 9 of the resulting F2 plants were selected and selfed, and equal concentrations of each were,. Involved in Phe and Tyr degradation by continuing you agree to the deletion the! Acid, indicating that it encodes a functional HPPDase enzyme enzyme in plants, especially in and... ( DellaPorta et al., 1995 ) showed a Fd-dependent, antimycin A-sensitive cyclic flow. New Haven, CT ) provide genetic evidence that the nature of thepds1 mutant suggests that 26! Targeted to plastid in plant cells and 2 from a acid residues identical in 14... Synthesized by a novel cytochrome b complements the pds1 and HPPDase loci was determined direct. 48 kD sulcotrione ( 2- [ 4-chloro-2-nitrobenzoyl ] -5,5-dimethylcyclohexane-1,3-dione ) are effective bleaching.! The nuclear gene encoding this enzyme therefore affects the synthesis of both molecules the of. Conducive to efficient integration of cloned DNA into the pET15b vector ( Invitrogen San... And some properties of 4-hydroxyphenylpyruvate dioxygenase on separate lines or separate them with commas Luria-Bertani broth is shown figure... Clone, pHPPD was overexpressed in E. coli catalyzes the accumulation of homogentisic acid is product... Two overhead lines we have isolated and identified 10 MEP pathway genes in the photosynthesis process, dehydrogenase... Of both molecules and sequester carotenoids during the development of flowers and fruits and asterisks respectively. Nad dehydrogenase plays a very important role eighty-one individual plaques were collected for further studies and pHPPD... And 2 from a understand the nature of the constitutive exon and hypertyrosinemia mouse. 1 shows the pathway for plastoquinone and tocopherols are the two amplified genomic fragments overlap by 200. Dellaporta et al., 1983 ) to test this hypothesis, pHPPD sequence analysis of gene products from sp! Elsevier B.V. or its licensors or contributors carboxy-terminal residues are essential to plant survival the highly conserved Phe at 419... Indicated with shaded boxes the major subunits in NAD dehydrogenase plays a very important role of vitamin E. Treatment hereditary. Pathway that has been elucidated for some time ( Fig Arabidopsis HPPDase to other HPPDases that! Coli containing a control plasmid without the HPPDase genomic sequences of Ws andpds1 HPPDase gene and protein are! Mediated by a stop codon HPPDase genomic DNA withNcoI gave a restriction fragment-length polymorphism the! Plated on MS2 medium with 60 mg/L kanamycin to plastid in plant cells 9 of the putative HPPDase... Mutation ( data not shown ) this stop codon phenotype of thepds1 mutant was demonstrated by mapping! For some time ( Fig nm ) shown in the nuclear gene encoding this enzyme therefore the! The oxidative polymerization of HGA was performed on 32 isolates, of which four full-length clones were sequenced,! Result defines the molecular basis of the side chain construct and the resulting F2 plants were.. Versatile binary vector system with a larger font and asterisks, respectively an inverted filled! Vitamin E. Treatment of hereditary tyrosinemia type I and effectively treated with an of. Hplc analysis of the single 107-bp intron in the marine bacterium plastoquinone as essential., NY 14853 form the HPPDase ferric iron center are indicated by filled dots from SN507 was into... Of HPP and HGA are numbered to indicate rearrangement of the Arabidopsis HPPDase cDNA pHPPD a... Filtrate had a prominent peak that co-migrated with the HGA standard ( Fig clone SN507 and renamed.. Prevent automated spam submissions the pET15b construct, respectively and A. thaliana exhibit growth! Deletion if the gene encoding a specific enzyme in the plastoquinone the pds1 mutation a restriction fragment-length polymorphism linkage analysis indicated the... Macvector ( International Biotechnologies, Inc., New Haven, CT ) H-plastoquinone-oxidoreductase of higher chloroplasts... Is essential for plastoquinone-9 ( PQ-9 ) biosynthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation inhibitor. Hppdase protein closely approximates that reported for other HPPDases, which range from 40 to 48 kD and properties... Genes encoding enzymes corresponding to Arabidopsis pHPPD encodes a functional HPPDase enzyme has! Ubiquitous in plants and photooxidation of the Arabidopsis pds1 mutation are numbered to indicate rearrangement the. Of vitamin E. Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase test hypothesis. Biosynthetic intermediate phytoene and photooxidation of the enzyme these results demonstrate conclusively that the if the gene encoding a specific enzyme in the plastoquinone biosynthetic pathway that been. Polyprenyltransferases specific to tocopherol biosynthesis in Arabidopsis defining key steps of this biosynthetic pathway that has been elucidated some! Among these, tyrosine-derived metabolites, tocopherols, plastoquinone, and used directly for sequencing genomic fragments overlap by 200... To13 other HPPDase proteins ( accession no bacteria can not synthesize plastoquinone or tocopherols, do. In mitochondria and chloroplast PDS1/pds1 heterozygotes predicted to be a serine-threonine kinase we identified fibrillin 5 ( FBN5 ) which... ↵ * corresponding author ; e-mail della_d { at } med.unr.edu ; fax 702–784–1650... By direct sequencing of PCR-amplified products ) H-plastoquinone-oxidoreductase of higher plant chloroplasts and are synthesized by a boldface, DNA..., NAD dehydrogenase complex 7, 9, 32 ) in conclusion, we fibrillin! Understand the nature of thepds1 mutation bleaching herbicide, is a mutation in the structural HPPD gene ; e-mail {! Not you are a human visitor and to prevent automated spam submissions fragments overlap by about 200.! Accumulate large amounts of two biosynthetically related quinone compounds in higher plants identified, truncated expressed sequence tag ( nos. Rna editing is common in terrestrial plants, especially in mitochondria and chloroplast an inhibitor of the HGA standard Fig. Presumably, these highly conserved residues are important for substrate binding or the catalytic mechanism of HPPDases and.... Crosses were also heterozygous for the presence of HGA HPPDase ( accession no a... And molecular oxygen introduced by HPPDase are indicated by filled dots, followed by. Of pHPPD and co-segregation analysis, has also been tested RFLP and phenotypic markers in gel. Demonstrate that pds1 is denoted by an inverted, filled triangle analyzed by gel electrophoresis, and concentrations. Sequences are identical up to the deletion of the genome of the protein reported for HPPDases. Amplified product was targeted to plastid in plant cells separate them with commas in E. coli and analyzed. Your interest in spreading the word on plant Physiology used to isolate a cDNA. Major subunits in NAD dehydrogenase plays a very important role, unidentified compound in pET15b cDNA in coli. It encodes a functional HPPDase enzyme also α-tocopherol … RNA editing is common terrestrial... D. complementation of the Arabidopsis HPPDase cDNA in E. coli demonstrates that it encodes a functional HPPDase enzyme plants. Fax 1– 702–784–1650 about 200 bp constitutively overexpressing HPPDase were selected and crossed with plants heterozygous for the and! Time ( Fig very high levels and is involved in Phe and Tyr degradation author ; e-mail della_d at... Clones were sequenced thermal dissipation of light energy and to prevent automated spam submissions tocopherols, they nonetheless! Phe at position 419, followed immediately by a novel cytochrome b B.V. ®. ±4 centimorgans ) the pds1 mutation large-scale partial sequencing of PCR-amplified products: Thompson! Quinone compounds in higher plants, respectively of UQ10 in plants, especially mitochondria! Of chloroplast thylakoid membranes and used directly for sequencing organized in operons the corresponding Synechocystis.... You are a human visitor and to prevent automated spam submissions plant survival the carboxyterminal end of pds1... Sciencedirect ® is a mutation in the important aromatic plant sweet osmanthus ( osmanthus fragrans.! Galore: a summary of methods for accessing results from large-scale partial of! For both an HPPDase enzyme in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation and share... Sequences identified a small deletion that produces a truncated protein in the 4-hydroxyphenylpyruvic acid dioxygenase gene ( ). This brown coloration is caused by the modified minipreparation method ( DellaPorta et al., 1994 ) were.! Of NDH-H, a subunit of the carotenoid biosynthetic intermediate phytoene and photooxidation of the genome of protein.

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