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Plant Physiol. (1999) 119: 1507-1516 Characterization and Subcellular Compartmentation of Recombinant 4-Hydroxyphenylpyruvate Dioxygenase from Arabidopsis in Transgenic Tobacco1
Unité Mixte Centre National de la Recherche Scientifique/Rhône-Poulenc (Unité, Mixte de Recherche 41) (I.G., R.P., M.M.); Département des Biotechnologies, Rhône-Poulenc Agrochimie, 14-20 rue Pierre Baizet, 69263 Lyon cedex 09, France (M.R.); and Department of Biology, Texas A&M University, College Station, Texas 77843 (T.-F.H.)
4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) from p-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacterium tumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.
4HPPD (EC 1.13.11.27) catalyzes the formation of homogentisate
(2,5-dihydroxyphenylacetate) from 4HPP and molecular oxygen. This
reaction proceeds through an oxidative decarboxylation of the 2-oxoacid
side chain of the substrate, which is accompanied by hydroxylation of
the aromatic ring and a 1,2-migration of the carboxymethyl group
(Jefford and Cadby, 1981 In photosynthetic organisms, however, this enzyme plays a specific and
crucial role because the product of the reaction, homogentisate, is the
aromatic precursor of all plastoquinones and tocopherols, essential
elements of the photosynthetic electron transport chain and of the
antioxidative systems, respectively. Presumably, as in other organisms,
plant 4HPPD is also involved in the degradation of Tyr (Durand and
Zenk, 1974 To improve our knowledge of this herbicide target, we recently purified
to near homogeneity the 4HPPD of cultured carrot cells (Lenne et al.,
1995 We report here the molecular and biochemical characterization of an
Arabidopsis 4HPPD. We also examined the cellular compartmentation of
the recombinant Arabidopsis 4HPPD by overexpressing the complete coding
sequence in transgenic tobacco.
The accession number for the nucleotide sequence of the Arabidopsis
4HPPD cDNA is AF047834.
Isolation of a Full-Length 4HPPD cDNA
Cloning of the Arabidopsis 4HPPD Sequence into the Expression Vector pTrc 99A The pTrc 99A-AT4-4HPPD plasmid coding for an Arabidopsis 4HPPD protein was constructed via site-directed mutagenesis using PCR amplification of the entire Arabidopsis 4HPPD cDNA. The following oligonucleotides were used: P1 (5 -GTTGGTGAAATCCATGGGCCACCAAAACGCCG-3), which
introduces a NcoI restriction site containing the ATG
translation-initiation codon (underlined), and P2
(5-GTTGGTTCTTCGTCGACTTCATCCCAC-3), which is complementary
to the 3 end of the cDNA-coding region and introduces a
SalI restriction site (underlined) 1 bp after the TGA
stop codon. PCR was performed for 15 cycles, including 1 min of
denaturation at 95°C, 1 min of annealing at 58°C, and 2 min of DNA
elongation at 72°C, driven by the Pwo DNA polymerase (Boehringer
Mannheim). The PCR DNA fragment was subcloned into the
pTrc 99A vector (Pharmacia), which was digested by
NcoI-SalI restriction enzymes. This
oriented cloning put the Arabidopsis 4HPPD cDNA under the control of
the lacUV5 promoter. The DNA insert was sequenced on
both strands to ensure that no mutation had been introduced during the
course of PCR amplification. The plasmid was termed pTrc
99A-AT4-4HPPD.
Genomic Southern Analysis Total DNA was isolated from young Arabidopsis plants, and 4 to 8 µg was digested overnight with the appropriate restriction enzymes (New England Biolabs). Fragments were then separated by 0.8% (w/v) agarose gel electrophoresis for 6 to 8 h. After alkaline blotting to a Hybond-N+ membrane (Amersham), hybridization was carried out with the full-length cDNA or with the 5 or 3 end of
the full-length cDNA (BamHI-PvuI, 568 bp
for the 5 end and PvuI-XhoI, 959 bp for
the 3 end) that had been 32P-labeled with a random-priming
kit (Ready-to-Go, Pharmacia).
Northern Analysis mRNAs isolated from Arabidopsis seedlings using an mRNA-isolation system (Straight A, Novagen, Madison, WI) were denatured (2-4 µg, 1 h at 50°C in 10 mM NaPi, pH 7.0, 1.08 M glyoxal, 2% [v/v] DMSO), separated by 1% (w/v) agarose gel electrophoresis, and transferred to a nylon membrane (Nytran, Schleicher & Schuell) according to the method of Thomas (1980) . The resulting blots were subjected to hybridization with the
desired 32P-labeled cDNA probe.
Overproduction of Recombinant Arabidopsis 4HPPD Escherichia coli JM105 cells harboring the pTrc 99A-AT4-4HPPD plasmid were grown at 37°C in 1 L of Luria-Bertani broth supplemented with 100 µg mL 1
carbenicillin and 100 µg mL1 streptomycin
(Maniatis et al., 1982).
Isopropyl- -D-thiogalactoside was added to a
final concentration of 1 mM when bacterial growth was equivalent to an A600 of 0.6. The cells
were grown for another 16 h at 30°C. The cells were harvested by
centrifugation and the pellet was resuspended in 10 mL of buffer A (20 mM potassium phosphate, pH 6.8, 1 mM EDTA, 1 mM DTT, 5 mM 6-aminohexanoic acid, and 1 mM
benzamidine) and sonicated using a cell disrupter (Vibra-Cell, Sonics & Materials, Danbury, CT) (100 pulses every 3 s on a power setting
of 5). The crude extract was centrifuged at 35,000g for 30 min to yield a cell-free supernatant.
Electrophoretic Analyses of Proteins Proteins were separated by SDS-PAGE containing 12% (w/v) acrylamide. The experimental conditions for gel preparation, sample solubilization, electrophoresis, and gel staining were as detailed by Chua (1980). PAGE under nondenaturing conditions was carried out at
equilibrium in the absence of any denaturing agent (SDS or DTT), as
described by Lasky (1978), on a linear acrylamide gradient
(3.5%-27%) with a 3.5% acrylamide stacking gel.
Immunoblotting Analysis After separation by PAGE, the proteins were electrophoretically transferred onto nitrocellulose membranes (Bio-Rad) according to the method of Towbin et al. (1979). Membranes were incubated for 30 min in
TBS (10 mM Tris-HCl, pH 7.6, and 150 mM NaCl)
containing 2% (v/v) Tween 20. They were incubated for 2 h with
the specific antibodies, in TBS plus 0.05% (v/v) Tween 20, and 1 h with goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad).
Membranes were stained for peroxidase activity using chloronaphtol and
hydrogen peroxide as substrates. Western analyses of the subcellular
fractions of Arabidopsis cells were revealed by chemiluminescence
according to the manufacturer's protocol (Boehringer Mannheim).
Assay for 4HPPD HPLC Determination of Homogentisate Activity was measured in a 200-µL volume containing 100 mM Tris-HCl, pH 7.5, 50 mM ascorbate, and 200 µM 4HPP. The reaction medium was incubated for 15 min at 30°C, except when otherwise stated. The reaction was stopped by the addition of 70 µL of 20% (w/v) perchloric acid. The precipitated protein was removed by centrifugation at 15,000g for 5 min. The amount of homogentisate formed was determined by HPLC. An aliquot fraction (50-100 µL) of the perchloric acid supernatant was injected onto a pico-Tag C18 column (3.9 mm × 15 cm, 10-µm particle size; Millipore) connected to an HPLC system. The buffers used for elution were: buffer A, 0.1% (v/v) trifluoroacetic acid in distilled water, and buffer B, 0.07% (v/v) trifluoroacetic acid in 80% (v/v) CH3CN. The following linear gradients were used: 0% (100% buffer A) to 70% buffer B, 0 to 17 min; 70% to 100% buffer B, 17 to 20 min; 100% buffer B, 20 to 24 min; 100% to 0% buffer B, 24 to 28 min, flow rate 1 mL min1. We detected the homogentisate by
measuring the UV A288. We quantitated the
homogentisate by measuring peak areas with a chromatograph data system
(450-MT2 software, Kontron Instruments, Eching, Germany). Peak areas
were converted to nanomoles of homogentisate by comparison with a
standard curve.
Oxygen Consumption The activity of the recombinant Arabidopsis 4HPPD was followed by measuring the oxygen consumption with a Clark-type electrode (Hansatech, King's Lynn, Norfolk, UK). We measured the activity in 500 µL, containing 100 mM Tris-HCl, pH 7.5, 50 mM ascorbate, and 200 µM 4HPP, at 30°C.Transformation of Tobacco Plants Plasmid Constructions We used two different constructs: Ara5, which contained the complete coding sequence of the 4HPPD cDNA of Arabidopsis, and Ara9, which was a fusion between an OTP and the complete coding sequence of the 4HPPD cDNA of Arabidopsis. The coding sequence for the OTP was constructed from the coding sequence for the transit peptide (171 bp) of the Rubisco small subunit from sunflower (Waksman and Freyssinet, 1987), followed by the first 66 nucleotides coding for the mature part
of the Rubisco small subunit from maize. The latter sequence was
followed by the 150 nucleotides coding for the transit peptide of the
Rubisco small subunit from maize (Lebrun et al., 1987). This yielded a
NcoI-SphI fragment of 387 bp. The presence of the
first 66 nucleotides of the mature Rubisco improved the targeting, and
the second transit peptide prevented the production of a fusion
protein. Details of this OTP fragment have been published (Lebrun et
al., 1992).
Plant Transformation Plasmids pRPA 150 A 2-ARA5 and pRPA 150 A2-ARA9 were
transferred by the helper plasmid JC2073 (Lebrun et al., 1992) to the Agrobacterium tumefaciens EHA 101 (Sailland et al., 1996) by
triparental mating (Lebrun et al., 1992) and used for transformation of
tobacco (Nicotiana tabacum cv PBD6) by leaf-disc
transformation, according to the method of Horsh et al. (1985).
Transformed cells were selected on regenerating medium containing 0.3 mg L1 isoxafutole, a potent inhibitor of 4HPPD
(Luscombe et al., 1995), for at least 5 weeks. In the presence of this
4HPPD inhibitor, untransformed cells developed white callus, whereas
transformed cells developed green callus as a consequence of the
overexpression of Arabidopsis 4HPPD (Sailland et al., 1996). The
presence of the transgene was confirmed by PCR and western analyses.
Immunocytochemical Localization of 4HPPD in Transformed Tobacco Plants Samples were dissected from the median part of young, subapical tobacco leaves in a drop of fixative solution and then immersion fixed for 3 h at room temperature in 2.5% paraformaldehyde and 0.1% glutaraldehyde in 0.05 M citrate phosphate buffer, pH 7.0. Leaf samples were subsequently rinsed in 0.1 M buffer, dehydrated in ethanol up to 70%, and then embedded in London White resin in gelatin capsules. Polymerization was carried out for 22 h at 56°C. Cross-sections, 80 nm thick, were cut from the blocks with a diamond knife and harvested on 300-mesh, Formvar-coated nickel grids. The grids were successively floated for 1 h on a blocking solution of 3% BSA and 0.01% Tween 20 in 0.05 M TBS and then twice for 5 min on a dilution buffer composed of 0.3% BSA and 0.01% Tween 20 in 0.05 M TBS. The primary antibody reaction for 4HPPD detection was performed in a 1:5 dilution of affinity-purified rabbit anti-4HPPD serum in a dilution buffer. This step was omitted in the controls. Secondary antibody reaction was made by floating the grids on drops of a 1:50 dilution of the commercial gold-conjugated goat anti-rabbit serum (British Biocell International, Cardiff, UK). Two gold particle sizes were used: 15 nm, which was directly viewed, and 10 nm, which was subsequently amplified with a silver-enhancing kit (British Biocell International). In both cases, grids were floated for 20 min on a 3% (w/v) aqueous solution of uranyl acetate for additional contrast. Control and labeled sections were viewed at 80 kV on an electron microscope (model CM10, Philips, Eindhoven, The Netherlands) at the Electron Microscopy Center for Biology and Geology at the University of Lyon (France).Preparation and Fractionation of Arabidopsis Cell Protoplasts Preparation of protoplasts by enzymic digestion of 7-d-old cell cultures of Arabidopsis cell T87 (Axelos et al., 1992) and measurement
of marker enzyme activities were carried out as described previously
for carrot cell protoplasts (Garcia et al., 1997).
Isolation of cDNA Clones Encoding Arabidopsis 4HPPD To characterize the Arabidopsis 4HPPD at the molecular level, we isolated a full-length 4HPPD cDNA by screening a cDNA library of young Arabidopsis leaves with an Arabidopsis expressed sequence tag as a nucleotide probe (accession no. T20952). The predicted polypeptide encoded by this 4HPPD clone is presented in Figure 1 and matches closely that of the carrot 4HPPD (Garcia et al., 1997).
Southern and Northern Analyses Southern analysis was used to examine the number of copies of the gene encoding the Arabidopsis 4HPPD cDNA. Total DNA was digested with restriction enzymes that cut once (PvuI) or did not cut (BamHI, EcoRV, and HindIII) within the cDNA; the DNA was then double digested with the following enzyme combinations: PvuI-BamHI, PvuI-HindIII, and EcoRV-HindIII. As shown in Figure 2, hybridization of the DNA digested with BamHI, EcoRV, or HindIII with the full-length probe produced one band of 23.0, 9.4, and 3.9 kb, respectively. Digestion with PvuI resulted in two bands (3.0 and 25 kb). When the DNA was double digested by PvuI-BamHI, two bands of 3.0 and 12.6 kb were revealed. The double digestion by PvuI-HindIII revealed three bands of 3.9, 2.5, and 1.6 kb. These results, along with those obtained by hybridization of the 5 and 3 ends of the probe, and
the fact that Southern hybridization carried out at the lower temperature of 50°C did not change the pattern of hybridization, strongly suggest the existence of a single copy of the gene encoding this 4HPPD in Arabidopsis.
Functional Characterization of the Recombinant 4HPPD
Subcellular Localization of the Recombinant 4HPPD
Immunolocalization of the Recombinant 4HPPD in Transformed Tobacco Leaves 4HPPD was immunodetected by western analyses of protein extracts from purified chloroplasts and from total soluble leaf extract. In denaturating conditions the large subunit of Rubisco, the most abundant protein of green tissue, had a molecular mass close to that of 4HPPD (52 and 49 kD, respectively), and its migration during SDS-PAGE interfered with that of 4HPPD, thereby preventing its accurate immunodetection. Under native conditions, the recombinant Arabidopsis 4HPPD migrated as a 100-kD polypeptide (Fig. 6), confirming that, as with the carrot enzyme (Garcia et al., 1997), it behaved as an homodimer. In these
conditions the recombinant 4HPPD was well separated from Rubisco, which
had a native molecular mass in excess of 500 kD. Protein extracts were
separated by nondenaturating electrophoresis prior to western analysis
using immunopurified anti-carrot 4HPPD antibody.
Immunolocalization of 4HPPD by Western Analyses of Cultured
Arabidopsis Cells
Plant 4HPPD is an intriguing enzyme because it is involved in two
distinct processes: the catabolism of Tyr and the biosynthesis of
prenylquinones. It was first assumed that the substrate of this enzyme
activity, 4HPP, was a chloroplastic intermediate in the shikimate
biosynthetic pathway leading to Tyr. However, it is now well documented
that arogenate appears to be the exclusive precursor of
L-Tyr in most plant species, including tobacco and spinach
(Jung et al., 1986). Therefore, 4HPP and, consequently, homogentisate
derive exclusively from the degradation of Tyr. Homogentisate may
either enter the prenylquinone biosynthesis pathway or be metabolized
by homogentisate dioxygenase (Tyr catabolism). To regulate the flux of
homogentisate between these two processes, one might postulate the
involvement of distinct isoforms of 4HPPD. Studies using spinach
(Fiedler et al., 1982 Received September 10, 1998;
accepted January 4, 1999.
Abbreviations:
4HPP, 4-hydroxyphenylpyruvate.
4HPPD, 4HPP
dioxygenase.
OTP, optimized transit peptide.
We are grateful to Roland Douce and Dominique Job for invaluable
scientific discussions and critical reading of the manuscript, to Anne
Roland for help in the buildup of the fusion OTP-4HPPD, to Martine
Freyssinet for assistance in tobacco transformation, and to Claudette
Job for her help in photograph reproduction.
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Abstract
Introduction
ZAPII (Stratagene). Plaque screening was
performed according to the manufacturer's instructions. Approximately
250,000 clones were screened, yielding six positive cDNA clones. We
analyzed the clone containing the longest insert and carried out DNA
sequence analysis on both strands, using a kit (PRISM, Applied
Biosystems) with fluorescent dideoxynucleotides, Taq DNA
polymerase, and T3 and T7 universal primers. Specific oligonucleotide
primers were used for further sequencing. The programs, Gene Works 5.2 (Oxford Molecular Group, Oxford, UK) and PCGENE (Intelligenetics,
Oxford Molecular Group), performed the sequence analyses.
![V=<FR><NU>V<SUB><UP>max</UP></SUB><UP> [4HPP]</UP></NU><DE>K<SUP>4HPP</SUP><SUB><UP>max</UP></SUB><UP>+[4HPP]</UP></DE></FR>](/content/vol119/issue4/fulltext/1507/img001.gif)
, Mes 100 mM; 
, Tris-HCl 100 mM.
10 nm, 1/50, amplification 2 min). A very intense signal
was specifically revealed within the chloroplasts. B, Transgenic
tobacco transformed by the construct ARA5 (×5,700); spongy mesophyll
(AB1 1/5, AB2-gold 
