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Plant Physiol. (1998) 117: 1317-1323
Complementation of the Arabidopsis pds1 Mutation with
the Gene Encoding p-Hydroxyphenylpyruvate Dioxygenase
Susan R. Norris1,
Xiaohua Shen, and
Dean Della Penna*
Departments of Biochemistry and Plant Sciences, University of
Arizona, Tucson, Arizona 85721 (S.R.N.); and Department of
Biochemistry/200, University of Nevada, Reno, Nevada 89557 (X.S.,
D.D.P.)
 |
ABSTRACT |
Plastoquinone and tocopherols are the
two major quinone compounds in higher plant chloroplasts and are
synthesized by a common pathway. In previous studies we characterized
two loci in Arabidopsis defining key steps of this biosynthetic
pathway. Mutation of the PDS1 locus disrupts the
activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both
plastoquinone and tocopherols in plants. Although plants homozygous for
the pds1 mutation could be rescued by growth in the
presence of homogentisic acid, the product of HPPDase, we were unable
to determine if the mutation directly or indirectly disrupted HPPDase
activity. This paper reports the isolation of a cDNA, pHPPD, encoding
Arabidopsis HPPDase and its functional characterization by expression
in both plants and Escherichia coli. 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. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid,
indicating that it encodes a functional HPPDase enzyme. Mapping of
pHPPD and co-segregation analysis of the pds1 mutation
and the HPPD gene indicate tight linkage. Constitutive expression of
pHPPD in a pds1 mutant background complements this
mutation. Finally, comparison of the HPPD genomic sequences from wild
type and pds1 identified a 17-bp deletion in the
pds1 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.
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INTRODUCTION |
Plastoquinone and tocopherols are the two major classes of
chloroplastic, lipid-soluble quinone compounds in higher plants. Plastoquinone is best known for its role as an electron carrier between
PSII and the Cyt b6/f complex, and to
a lesser extent as an electron carrier for NAD(P)H-plastoquinone
oxidoreductases (Berger et al., 1993 ). In mammals, which cannot
synthesize plastoquinone or tocopherols,  -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, see
Liebler, 1993). 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).
Figure 1 shows the pathway for
plastoquinone and tocopherol biosynthesis in plants. 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). HPPDase catalyzes a complex, irreversible reaction
involving the introduction of two molecules of oxygen, and
decarboxylation and rearrangement of the side chain (Fig. 1). 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).
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| Figure 1.
The plastoquinone and -tocopherol biosynthetic
pathway in higher plants. For clarity, not all biosynthetic steps are
shown and only the HPPDase reaction is shown in detail. The
CO2 lost and molecular oxygen introduced by HPPDase are
indicated with a larger font and asterisks, respectively. The
conjugated rings of HPP and HGA are numbered to indicate rearrangement
of the side chain. The locations of the pds1 and
pds2 mutations in the pathway are indicated.
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Although mammals and nonphotosynthetic bacteria cannot synthesize
plastoquinone or tocopherols, they do nonetheless contain HPPDase
enzymatic activity. This activity is often present at very high levels
and is involved in Phe and Tyr degradation. 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.
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). 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. 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. In plants, triketones such as
sulcotrione (2-[4-chloro-2-nitrobenzoyl]-5,5-dimethylcyclohexane-1,3-dione) are
effective bleaching herbicides. 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). The latter results in accumulation
of the carotenoid biosynthetic intermediate phytoene and photooxidation
of the plastid.
A genetic basis for the effects of triketones on plant carotenoid
synthesis was suggested by the identification of two Arabidopsis mutations that disrupt phytoene
desaturation (pds mutations) but do
not map to the phytoene desaturase enzyme locus (Norris et al., 1995).
Previous work demonstrated that mutations in either the PDS1
or 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). The pds mutations thus provide genetic evidence that
plastoquinone is an essential component for carotenoid biosynthesis in
plants and provide insight into plastidic quinone synthesis and
function. 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. 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).
To functionally test the hypothesis that the Arabidopsis
pds1 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. 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 in Escherichia coli. Linkage of the HPPD gene and the
pds1 mutant was demonstrated by both mapping and
co-segregation analysis. Sequence analysis of the wild-type and mutant
HPPDase genomic sequences identified a small deletion that produces a
truncated protein in the mutant. Finally, we show functional
complementation of the pds1 mutant phenotype when the
HPPDase cDNA is constitutively expressed. Combined, these data
conclusively demonstrate that pds1 is a mutation of the
HPPDase gene.
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MATERIALS AND METHODS |
cDNA and Genomic DNA Isolation and DNA Sequence Analysis
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. T20952) with
homology to the carboxy terminus of human HPPDase (accession no.
X72389). The 460-bp insert from this expressed sequence tag was used as
a probe to screen 4 × 105 plaques of the
Arabidopsis PRL2 library (Newman et al., 1994). 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. Isolate 18 was chosen for further
studies and renamed pHPPD. A pHPPD probe was made by labeling a
SalI/NotI fragment of pHPPD using the Random
Prime kit (Boehringer Mannheim).
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). 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. 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. In both cases, the two amplified genomic fragments overlap
by about 200 bp.
Three independent sets of PCR reactions were performed for each
fragment amplification. PCR products were analyzed by gel electrophoresis, and equal concentrations of each were pooled, purified, and used directly for sequencing. DNA sequencing was performed using a dye deoxy terminator cycle sequencing kit (Applied Biosystems) and an automated DNA sequencer (model 310, Applied Biosystems). DNA-sequence analysis was done using both DNAStar and
MacVector (International Biotechnologies, Inc., New Haven, CT).
Protein Overexpression
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 amplified product was ligated into the pCRII vector (Invitrogen,
San Diego, CA), generating clone SN507. A 1.49-kb
NcoI/BamHI fragment from SN507 was ligated into
the pET15b vector (Novagen, Madison, WI), generating pET-HPPD. pET15b
and pET-HPPD were transformed into Escherichia coli cell
line BL21(DE3) (Novagen) via electroporation. HPLC analysis of
bacterial cultures for the presence of HGA was performed according to
published procedures (Denoya et al., 1994). 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.
Linkage Analysis
Co-segregation of the pds1 and HPPDase loci was
determined by restriction fragment-length polymorphism linkage analysis
using pHPPD as probe. F2 progeny heterozygous for
the pds1 mutation were selected from a cross between
PDS1/pds1 (ecotype Ws) and PDS1/PDS1 (ecotype
Columbia [Col]). Digestion of Ws and Col genomic DNA with
NcoI gave a restriction fragment-length polymorphism for the
pHPPD probe. Genomic DNA for co-segregation analysis was isolated from
F2 progeny by the modified minipreparation method (DellaPorta et al., 1983). The digested DNA was separated on a 0.6%
agarose gel and transferred to a nylon membrane (Micron Separations, Westborough, MA). 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.
Plant Transformation
Clone SN500 was generated by subcloning a 1.5-kb
KpnI/HindIII fragment containing the complete
coding region of pHPPD into the plant-transformation shuttle vector
pART7 (Gleave, 1992). After partial digestion of SN500 with
NotI, 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. SN506 was
electroporated into Agrobacterium tumefaciens strain C58 and
used to transform wild-type Arabidopsis (ecotype Ws) via vacuum
infiltration (Bent et al., 1994). Seed was collected from individual
T1 plants, surface sterilized, and plated on MS2
medium (Norris et al., 1995) with 100 mg/L carbenicillin, 60 mg/L
kanamycin, and 10 mg/L benomyl. Kanamycin-resistant
T2 seedlings were transferred to soil and grown
to maturity, and T3 seed was harvested. For
complementation analysis, kanamycin-resistant T2
plants were crossed with PDS1/pds1 heterozygotes. The
resulting F1 seeds were surface sterilized and
plated on MS2 medium with 60 mg/L kanamycin. Kanamycin-resistant
F1 seedlings were transferred to soil and grown
as described above. 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 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.
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RESULTS |
Isolation and Characterization of an Arabidopsis HPPDase cDNA
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. 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.
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. This partial cDNA
was used as a probe to isolate a full-length cDNA that was named pHPPD.
The first ATG of pHPPD begins an open-reading frame encoding a 50-kD
protein of 445 amino acids (Fig. 2). The putative Arabidopsis
HPPDase protein has from 17% to 27% amino acid identity with
bacterial, fungal, and animal HPPDases and between 58% and 70%
amino acid identity with two other plant HPPDases. The estimated 50-kD
size of the Arabidopsis HPPDase protein closely approximates that
reported for other HPPDases, which range from 40 to 48 kD.
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| Figure 2.
Nucleotide and deduced amino acid sequence of the
Arabidopsis HPPDase cDNA pHPPD. The protein sequence is shown in
boldface underneath the nucleotide sequence (accession no. AF000228).
The nucleotide sequence of the originally identified, truncated
expressed sequence tag (accession no. T20952) is indicated by a single
underline. Alignments were performed to13 other HPPDase proteins
(accession nos. AJ000693, D64004, L38493, U11864, U87257, S69666,
M59289, M59429, Z50016, X72389, D29987, M18405, and D13390).
Arabidopsis HPPDase amino acid residues showing identity in 9 of the
other 13 HPPDase proteins are indicated with shaded boxes. Amino acid
residues identical in all 14 HPPDase sequences are denoted with black
boxes. The five conserved Tyr and His residues postulated to form the
HPPDase ferric iron center are indicated by filled dots. The location
of the single 107-bp intron in the HPPDase genomic sequences of Ws and
pds1 is denoted by an inverted, filled triangle. The
17-bp deletion in the HPPDase gene in pds1 is denoted by
a boldface, italic DNA sequence and two overhead lines. The Ws and
pds1 HPPDase gene and protein sequences are identical up
to the deletion. The consequence of this mutation at the protein level
is indicated in the box below the deletion.
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Functional Analysis of the Arabidopsis HPPDase Protein
The protein-sequence homology of the putative Arabidopsis HPPDase
to other HPPDases suggested that it encodes an HPPDase enzyme. To test
this hypothesis, pHPPD was overexpressed in E. coli and functionally analyzed. E. coli harboring the pET-HPPD
construct developed a dark-brown color, whereas cultures containing the empty pET15b vector did not (data not shown). A similar dark-brown coloration was reported when the gene encoding HPPDase from
Streptomyces avermitilis was expressed in E. coli
(Denoya et al., 1994). This brown coloration is caused by the
accumulation of ochronotic pigment, which forms upon the oxidative
polymerization of HGA. 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. 3). A HGA standard eluted at 7.9 min and had the spectra and absorbance maximum
(291 nm) shown in Figure 3B. The pET-HPPD culture filtrate had a
prominent peak that co-migrated with the HGA standard (Fig. 3A) and had
a spectrum and absorbance maximum that were identical to those of the
HGA standard (Fig. 3B). 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). These results indicate that Arabidopsis pHPPD encodes a
functional HPPDase enzyme.
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| Figure 3.
Expression of Arabidopsis HPPDase cDNA in
E. coli. A, HPLC analysis of a HGA standard in
Luria-Bertani broth is shown in the top plot. The middle and bottom
plots are cell-free extracts from cultures of E. coli
harboring the pET-HPPD construct and the pET15b construct,
respectively. B, Absorption spectra of peaks 1 and 2 from A. Peak 1, HGA standard and co-migrating peak in medium of pET-HPPD; peak 2, unidentified compound in pET15b.
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Mapping, Molecular Complementation, and Genomic Sequence Analysis
In previous work we demonstrated that the biochemical basis of the
Arabidopsis pds1 mutation is an inability to convert HPP to
HGA (Fig. 1; Norris et al., 1995). The PDS1 gene product
could therefore be the HPPDase enzyme, a regulator of HPPDase
expression or activity, or a cofactor required for HPPDase activity.
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 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). For finer mapping, segregation analysis of the pds1
mutation 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). Together, these data indicate that the PDS1 locus
and HPPDase gene are linked in the Arabidopsis genome.
Molecular complementation of the pds1 mutation with the
Arabidopsis pHPPD cDNA was undertaken to determine if the
pds1 mutant could be rescued by constitutive overexpression
of the wild-type HPPDase protein. A transcriptional fusion of the
cauliflower mosaic virus 35S promoter and the full-length pHPPD cDNA in
the sense orientation was used to transform wild-type Arabidopsis (Ws)
plants. Three independent transgenic lines constitutively
overexpressing HPPDase were selected and crossed with
PDS1/pds1 heterozygotes. Fifty percent of the resulting
kanamycin-resistant F1 progeny from these crosses
were also heterozygous for the pds1 mutation. 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 (Table
I).  2 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. Loss of
the transgene should restore a 3:1 green:white ratio to such plants.
This hypothesis was verified by analyzing the F1
plants that were 100% kanamycin sensitive; one-half of which
contained F2 progeny segregating 100% green
(PDS1/PDS1) and half of which segregated 3:1 green:white
(PDS1/pds1) (data not shown). These data demonstrate that
overexpression of a wild-type HPPDase protein in the pds1
mutant background complements the mutation and suggest that the
molecular basis of the pds1 mutation is a disruption in the
HPPDase gene.
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Table I.
Segregation analysis of progeny from plants
heterozygous for both an HPPDase transgene and the pds1 mutation
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To determine the molecular basis of the pds1 mutation, the
HPPDase genomic DNA sequences from wild-type Ws Arabidopsis (accession no. AF060481) and pds1 mutant tissues were determined by
direct sequencing of PCR-amplified products. Both HPPDase genomic
sequences contain a single 107-bp intron of identical sequence between
positions 1162 and 1163 of the HPPD cDNA sequence in Figure 2. 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.
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. 2). This stop codon results in the deletion of the remaining 26 amino acids from the carboxyterminal end of the protein. This result
defines the molecular basis of the pds1 mutation as a
mutation in the structural HPPD gene.
| |
DISCUSSION |
The plastids of higher plants accumulate large amounts of two
biosynthetically related quinone compounds: plastoquinones and tocopherols. Plastoquinones are fundamentally important components of
the photosynthetic electron-transport chain, whereas tocopherols are
thought to be important for free radical scavenging and protection from
oxidative stress. Plastoquinone and tocopherols share a common biosynthetic pathway that has been elucidated for some time (Fig. 1).
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). One of these mutations,
pds1, was shown to affect the activity of HPPDase, the
committed step of plastidic quinone biosynthesis (Fig. 1). To further
understand the nature of the pds1 mutation, we have isolated
and functionally analyzed cDNAs and genomic clones encoding HPPDase
from Arabidopsis.
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. This expressed sequence tag was used to isolate a full-length
Arabidopsis cDNA clone, pHPPD. 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. 2).
Presumably, these highly conserved residues are important for substrate
binding or the catalytic mechanism of HPPDases. 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. 2).
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. 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. 3). E. coli containing a control plasmid without the
HPPDase open-reading frame lacks this peak (Fig. 3A). In addition to
HPPDase-dependent HGA accumulation, pHPPD expression in E. coli resulted in accumulation of ochronotic pigment, an oxidative
polymerization product of HGA. Similar results were reported when a
S. avermitilis HPPDase was expressed in E. coli
(Denoya et al., 1994). These data demonstrate that the Arabidopsis cDNA
pHPPD encodes a functional HPPDase enzyme.
As discussed previously, Arabidopsis plants homozygous for the
pds1 mutation are unable to synthesize both plastoquinone
and tocopherols because of an inability to convert HPP to HGA (Fig. 1).
Although it was clear from previous work that the pds1
mutation affected HPPDase activity, we could not determine whether the pds1 mutation directly or indirectly affected the HPPDase
enzyme (Norris et al., 1995). 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.
Linkage analysis indicated that the gene corresponding to Arabidopsis
pHPPD maps near (±4 centimorgans) the pds1 mutation (data
not shown). Transgenic plants overexpressing the pHPPD cDNA were
generated in a wild-type background and crossed with plants heterozygous for the pds1 mutation. Kanamycin-resistant
F1 plants were selected and selfed, and the
resulting F2 plants were scored. 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 the
pds1 mutation were not linked. 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. These
data provide genetic evidence that constitutive expression of the pHPPD transgene complements the pds1 mutation.
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. 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 pds1
HPPD allele. This deletion results in a substitution of Leu for the
highly conserved Phe at position 419, followed immediately by a stop
codon. 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. The null phenotype of the
pds1 mutant suggests that these 26 carboxy-terminal residues
are essential for HPPDase enzymatic activity. Most significantly, these
data define the molecular basis of the pds1 mutation as a
lesion in the structural HPPD gene.
In conclusion, we have identified and characterized Arabidopsis cDNA
and genomic clones encoding HPPDase. The functional expression of the
Arabidopsis HPPDase cDNA in E. coli demonstrates that it encodes a functional HPPDase enzyme. Constitutive expression of the
protein encoded by the Arabidopsis HPPDase cDNA is sufficient to
restore wild-type pigmentation to plants homozygous for the pds1 mutation. 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. These results demonstrate conclusively that the nature of the
pds1 mutation in Arabidopsis is a mutation in the gene encoding the HPPDase enzyme. Future studies will determine the consequences of overexpressing the wild-type HPPDase enzyme in plants.
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.
| |
FOOTNOTES |
1
Present address: Boyce Thompson Institute, Tower
Road, Ithaca, NY 14853.
*
Corresponding author; e-mail della_d{at}med.unr.edu; fax
1- 702-784-1650.
Received January 12, 1998;
accepted May 4, 1998.
| |
ABBREVIATIONS |
Abbreviations:
HGA, homogentisic acid.
HPP, p-hydroxyphenylpyruvate.
HPPDase, p-hydroxyphenylpyruvate dioxygenase.
| |
LITERATURE CITED |
Altschul SF,
Gish W,
Miller W,
Myers E,
Lipman DJ
(1990)
Basic local alignment search tool.
J Mol Biol
215:
403-410
[CrossRef][Web of Science][Medline]
Bent A,
Kunkel BN,
Dahlbeck D,
Brown KL,
Schmidt R,
Giraudat J,
Leung J,
Staskawicz BJ
(1994)
RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes.
Science
265:
1856-1860
[Abstract/Free Full Text]
Berger S,
Ellersiek U,
Westoff P,
Steinmuller K
(1993)
Studies on the expression of NDH-H, a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher plant chloroplasts.
Planta
190:
25-31
Coon SL,
Kotob S,
Jarvis BB,
Wang S,
Fuqua WC,
Weiner RM
(1994)
Homogentisic acid is the product of MelA, which mediates melanogenesis in the marine bacterium Shewanella colwelliana D.
Appl Environ Microbiol
60:
3006-3010
[Abstract/Free Full Text]
DellaPorta S,
Wood J,
Hicks J
(1983)
A plant DNA minipreparation: version 2.
Plant Mol Biol Rep
1:
19-22
Denoya CD,
Skinner DD,
Morgenstern MR
(1994)
A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.
J Bacteriol
176:
5312-5319
[Abstract/Free Full Text]
Endo F,
Awata H,
Katoh H,
Matsuda I
(1995)
A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III.
Genomics
25:
164-169
[CrossRef][Medline]
Endo F,
Awata H,
Tanoue A,
Ishiguro M,
Eda Y,
Titani K,
Matsuda I
(1992)
Primary structure deduced from complementary DNA sequence and expression in cultured cells of mammalian 4-hydroxyphenylpyruvic acid dioxygenase.
J Biol Chem
267:
24235-24240
[Abstract/Free Full Text]
Garcia I,
Rodgers M,
Lenne C,
Rolland A,
Sailland A,
Matringe M
(1997)
Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA.
Biochem J
325:
761-769
Gershwin ME,
Coppel RL,
Bearer E,
Peterson MG,
Sturgess A,
MacKay IR
(1987)
Molecular cloning of the liver-specific rat F antigen.
J Immunol
139:
3828-3833
[Abstract]
Gibbs TC,
Payan J,
Brett EM,
Lindstedt S,
Holme E,
Clayton PT
(1993)
Peripheral neuropathy as the presenting feature of tyrosinaemia type I and effectively treated with an inhibitor of 4-hydroxyphenylpyruvate dioxygenase.
J Neurol Neurosurg Psychiatry
56:
1129-1132
[Abstract/Free Full Text]
Gleave AP
(1992)
A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome.
Plant Mol Biol
20:
1203-1207
[CrossRef][Web of Science][Medline]
Hess JL
(1993)
Vitamin E, -tocopherol.
In
R Alscher,
J Hess,
eds, Antioxidants in Higher Plants.
CRC Press, Boca Raton, FL, pp 111-134
Hummel R,
Norgaard P,
Andreasen PH,
Neve S,
Skjodt K,
Tornehave D,
Kristiansen K
(1992)
Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with the membranes and Golgi apparatus and transport vesicles.
J Mol Biol
228:
850-861
[Medline]
Kaneko T,
Tanaka A,
Sato S,
Kotani H,
Sazuka T,
Miyajima N,
Sugiura M,
Tabata S
(1995)
Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features in the 1 Mb region from map positions 64% to 92% of the genome.
DNA Res
2:
153-166
[Abstract]
Liebler DC
(1993)
The role of metabolism in the antioxidant function of vitamin E.
Crit Rev Toxicol
23:
147-169
[Web of Science][Medline]
Lindstedt S,
Holome E,
Lock EA,
Hjalmarson O,
Strandvik B
(1992)
Treatment of hereditary tyrosinemia type I by inhibition of 4 hydroxyphenylpyruvate dioxygenase.
Lancet
340:
813-817
[CrossRef][Web of Science][Medline]
Lindstedt S,
Odelhog B,
Rundgren M
(1977)
Purification and some properties of 4-hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874.
Biochemistry
16:
3369-3377
[CrossRef][Medline]
Lister C,
Dean C
(1993)
Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana.
Plant J
4:
745-750
[CrossRef][Web of Science]
Mason K (1980) The first two decades of vitamin E history.
In LJ Machlin, ed, Vitamin E: A Comprehensive Treatise.
Marcel Dekker, New York, pp 1-6
Mayonado DJ,
Hatzios KK,
Orcutt DM,
Wilson HP
(1989)
Evaluation of the mechanism of action of the bleaching herbicide SC-0051 by HPLC analysis.
Pestic Biochem Physiol
35:
138-145
Newman T,
De Bruijn,
FJ,
Green P,
Keegstra K,
Kende H,
McIntosh L,
Ohlrogge J,
Raikhel N,
Somerville S,
Thomashow M,
and others
(1994)
Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones.
Plant Physiol
106:
1241-1255
[Abstract]
Norris SR,
Barrette TR,
DellaPenna D
(1995)
Genetic dissection of carotenoid synthesis in Arabidopsis defines plastoquinone as an essential component of phytoene desaturation.
Plant Cell
7:
2139-2148
[Abstract]
Roche PA,
Moorehead TJ,
Hamilton GA
(1982)
Purification and properties of hog liver 4-hydroxyphenylpyruvate dioxygenase.
Arch Biochem Biophys
216:
62-73
[Medline]
Ruetschi U,
Dellsen A,
Sahlin P,
Stenman G,
Rymo L,
Lindstedt S
(1993)
Human 4 hydroxyphenylpyruvate dioxygenase: primary structure and chromosomal localization of the gene.
Eur J Biochem
213:
1081-1089
[Medline]
Schultz A,
Ort O,
Beyer P,
Kleinig H
(1993)
SC-0051, a 2-benzoylcyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase.
FEBS Lett
318:
162-166
[CrossRef][Medline]
Secor J
(1994)
Inhibition of barnyardgrass 4-hydroxyphenylpyruvate dioxygenase by sulcotrione.
Plant Physiol
106:
1429-1433
[Abstract]
Wada GH,
Fellman JH,
Fujita TS,
Roth ES
(1975)
Purification and properties of avian liver p-hydroxyphenylpyruvate hydroxylase.
J Biol Chem
250:
6720-6726
[Abstract/Free Full Text]
Wilson R,
Ainscough R,
Anderson K,
Baynes C,
Berks M,
Bonfield J,
Burton J,
Connell M,
Copsey T,
Cooper J,
and others
(1994)
2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans.
Nature
368:
32-38
[CrossRef][Medline]
Wintermeyer E,
Fluegel M,
Ott M,
Steinert M,
Rdest U,
Mann KH,
Hacker J
(1994)
Sequence determination and mutational analysis of the lly locus of Legionella pneumophila.
Infect Immun
62:
1109-1117
[Abstract/Free Full Text]
Wyckoff EE,
Pishko EJ,
Kirkland TN,
Cole GT
(1995)
Cloning and expression of a gene encoding a T-cell reactive protein from Coccidioides immitis: homology to a 4-hydroxyphenylpyruvate dioxygenase and the mammalian F antigen.
Gene
161:
107-111
[CrossRef][Web of Science][Medline]
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|
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|
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|
|
|
|
|
|
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|
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|
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Abstract
Introduction