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Plant Physiol. (1998) 117: 1227-1234
Identification of a Functional Homolog of the Yeast Copper
Homeostasis Gene ATX1 from
Arabidopsis1
Edward Himelblau2,
Helena Mira2,
Su-Ju Lin,
Valeria Cizewski Culotta,
Lola Peñarrubia, and
Richard M. Amasino*
Department of Biochemistry, University of Wisconsin, 420 Henry
Mall, Madison, Wisconsin 53706 (E.H., R.M.A.); Departament de
Bioquímica i Biologia Molecular, Universitat de València,
Dr. Moliner 50, Burjassot, València, E-46100 Spain (H.M., L.P.); and Division of Toxicological Sciences, Department of Environmental
Health Sciences, Johns Hopkins University School of Public Health,
Bethesda, Maryland 20892 (S.-J.L., V.C.C.)
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ABSTRACT |
A cDNA
clone encoding a homolog of the yeast (Saccharomyces
cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene,
referred to as Copper CHaperone
(CCH), encodes a protein that is 36% identical to the
amino acid sequence of ATX1 and has a 48-amino acid extension at
the C-terminal end, which is absent from ATX1 homologs
identified in animals. ATX1-deficient yeast
(atx1) displayed a loss of high-affinity iron uptake.
Expression of CCH in the atx1 strain
restored high-affinity iron uptake, demonstrating that
CCH is a functional homolog of ATX1. When
overexpressed in yeast lacking the superoxide dismutase gene
SOD1, both ATX1 and CCH
protected the cell from the reactive oxygen toxicity that results from
superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper,
indicating that CCH function is copper dependent. In
Arabidopsis CCH mRNA is present in the root, leaf, and
inflorescence and is up-regulated 7-fold in leaves undergoing
senescence. In plants treated with 800 nL/L ozone for 30 min,
CCH mRNA levels increased by 30%. In excised leaves and
whole plants treated with high levels of exogenous CuSO4,
CCH mRNA levels decreased, indicating that
CCH is regulated differently than characterized
metallothionein proteins in Arabidopsis.
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INTRODUCTION |
Copper, a plant micronutrient, acts as an effective electron
acceptor and donor in the active sites of many proteins involved in
oxidation and reduction reactions (for review, see Marschner, 1995 ).
These include electron-transport proteins such as Cyt oxidase and
proteins involved in the detoxification of oxygen radicals such as
copper/zinc SOD. A variety of enzymes with oxidase function, including
ascorbate oxidase, diamine oxidase, and phenol oxidase, require copper
for their activity.
Despite its importance to plant metabolism, copper is toxic at high
concentrations. Copper toxicity can be oxygen dependent through the
Haber-Weiss reaction, which generates reactive oxygen intermediates
(Halliwell and Gutteridge, 1989), or oxygen independent by
inappropriate binding to biomolecules (Kalstrom and Levine, 1991).
Organisms have evolved various metal homeostasis factors to control the
cellular accumulation, distribution, and sequestration of the metal
(Vulpe and Packman, 1995; Koch et al., 1997). In the yeast
Saccharomyces cerevisiae there is an overlap between systems
controlling copper-ion homeostasis and oxygen radical metabolism. In
yeast, copper-binding metallothioneins protect not only against copper
toxicity but also detoxify the superoxide anion (Tamai et al., 1993).
Similarly, the copper/zinc SOD1 contributes to both the maintenance of
copper homeostasis and to superoxide scavenging (Culotta et al., 1995).
The product of the yeast gene Anti-oxidant 1 (ATX1) shows a similar overlap between copper homeostasis
and oxygen radical metabolism. The ATX1 gene was originally
isolated in strains lacking SOD1 by its ability to suppress
oxygen toxicity in a copper-dependent manner (Lin and Culotta, 1995).
ATX1 encodes a soluble copper chaperone. ATX1 binds Cu(I) in
the cytoplasm and delivers it to a copper transporter in the membrane
of a post-Golgi vesicle. In the vesicle, the copper is inserted into a
multicopper oxidase essential for high-affinity iron uptake (Lin et
al., 1997; Pufahl et al., 1997). Thus, ATX1 may be involved in both
copper transport and defense against oxidative stress. HAH1, the human
homolog of ATX1, demonstrates both of these functions, with the amino acid residues involved in antioxidant function separate from the copper-binding region (Hung et al., 1998).
Here we describe the identification of the gene Copper
CHaperone (CCH) from Arabidopsis encoding a 121-amino
acid protein with sequence similarity to ATX1. By expressing
CCH in yeast, we show that CCH, like
ATX1, can protect SOD1-deficient yeast from
active oxygen toxicity. Moreover, CCH expression can also restore high-affinity iron uptake to ATX1-deficient yeast,
indicating that CCH and ATX1 are functional
homologs. Thus, the gene product of CCH may be involved in
both the detoxification of active oxygen and the delivery of copper.
Whereas there is basal CCH mRNA expression in many
Arabidopsis tissues, CCH mRNA levels increase during leaf senescence, suggesting a role for the CCH gene product in
that process.
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MATERIALS AND METHODS |
Plant Growth and Determination of Nutrient Content
Seedlings of Arabidopsis ecotype Columbia (Col) and Landsberg
erecta (Ler) were transplanted to a commercial potting mix
(Fafard Germination Mix, Agawam, MA) and grown at 24°C. Ler
plants were grown with continuous illumination of approximately 100 µmol/m2 of cool-white fluorescent light. Col
plants were grown with a photoperiod of 16 h of light/8 h of dark
in growth cabinets with 65 µmol/m2 of
cool-white fluorescent light.
Sequence Analysis
A partial amino acid sequence of SAM45 (Crowell and Amasino, 1991)
was entered into the database search program BLASTN (National Institutes of Health, Bethesda, MD). The Arabidopsis cDNA 31B12T7 (DBEST accession no. T04721; isolated by the Arabidopsis Expressed Sequence Tag Project Group [Newman et al., 1994]) revealed high homology to SAM45 and was ultimately named Copper CHaperone
(CCH). The nucleotide sequence of the CCH cDNA
clone was determined by cycle sequencing on an automated sequencer
(Applied Biosystems). The clone was sequenced completely from both the
5 and 3 ends. Sequences related to CCH were identified
using the program BLASTN. Alignments were generated by the Genetics
Computer Group software package (program BESTFIT, Madison, WI).
Predictions of secondary structure were determined using the
EMBL-Heidelberg data bank and the Genetics Computer Group software.
Yeast Strains and Vectors
The yeast (Saccharomyces cerevisiae) strains used in
this study were: KS107 (sod1 ) (Culotta et al., 1995) and
SL215 (atx1 derivative of YPH250) (Lin et al., 1997).
SL201 (sod1,ctr1) was constructed by
introducing a ctr1::LEU2 deletion
(Dancis et al., 1994) into KS107.
To create a vector to express CCH in yeast, the
CCH coding sequence was amplified from expressed sequence
tag cDNA clone 31B12T7 (Newman et al., 1994); the upstream primer for
amplification was designed such that an EcoRI site was
generated 3 bases upstream of the CCH start codon
(5-AAGAATTCGCCATGGCTCAGACCG-3). The downstream primer for
amplification was the SP6 primer (Promega). The 3 end of the amplified
fragment contained a BamHI site downstream of the
poly(A+) tail of the CCH cDNA. The
CCH coding region was directionally subcloned into the
EcoRI and BamHI sites of the yeast expression vector pSM703 (Culotta et al., 1995) to create vector pB-038 for CCH expression in yeast. Other plasmids used were p413-A1
(expression of ATX1) (Lin et al., 1997) and pRS413 (the
expression vector used to create p413-A1 with no insert) (Sikorski and
Hieter, 1989).
Complementation of Yeast by CCH
The atx1 strain SL215 was transformed with
p413-A1, pRS413, pSM703, or pB-038 as described previously (Lin et al.,
1997). To test for restoration of iron uptake in this strain,
transformants were grown on complete synthetic dextrose medium (Rose et
al., 1991) or synthetic dextrose medium buffered with 50 mM
Na-Mes, pH 6.1, and 3 mM ferrozine
(ferrozine(3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine); Sigma) with or without 50 µM
ferrous ammonium sulfate (Sigma) for 5 d at 30°C.
pB-038 and p413-A1 were transformed separately into KS107. To test for
reversal of Lys and Met auxotrophy of this SOD1-deficient yeast strain,
transformants were plated on complete synthetic dextrose medium,
synthetic dextrose medium without Lys, and synthetic dextrose medium
without Met, and grown in air for 3 d at 30°.
To determine whether CCH action was copper dependent, strain
KS107 was transformed with either pSM703 (an empty expression vector)
or pB-038 (a CCH expression vector). These were tested for oxygen
tolerance by aerobic growth in yeast extract-peptone-dextrose liquid medium as described previously (Lin and Culotta, 1995). Anaerobic growth was tested as described (Liu et al., 1992). Where indicated, cultures were supplemented with 150 µM BCS or
150 µM BCS plus 50 µM
CuSO4. After 18 h,
A600 was determined for each culture.
Ozone Treatment
Three- to 4-week-old plants (ecotype Col) grown under described
conditions were moved into 500-L ozone fumigation chambers located
inside growth cabinets. Ozone (800 nL/L) was generated by an ozonifier
(model Eco-Lab.ppm, Eco Ozono, Valencia, Spain) and monitored
continuously using an ozone analyzer (model 1180, Dabisi, Environment
Corp., Glendale, CA). Rosettes were harvested and frozen in
liquid nitrogen at the indicated times and stored at  80°C until RNA
isolation.
Metal Treatment
The fifth and sixth leaves of Arabidopsis were removed 16 DAG and
placed in Murashige and Skoog liquid medium (Murashige and Skoog, 1962)
with or without 50 µM
CuSO4·5H2O (Mallinckrodt,
Chesterfield, MO). Leaves were incubated in continuous light at
24°C for 18 h with gentle shaking. Total RNA was extracted from
the leaves as described below.
Three- to 4-week-old Arabidopsis plants (ecotype Col) were removed from
soil and their roots were submerged in 1 mM
CuSO4 solutions for 0.25, 0.5, 1, 2, 4, or 8 h. After treatment, shoots (primarily rosette tissue, no roots) were
harvested for nutrient analysis or for mRNA extraction. Copper uptake
was determined by atomic absorption spectroscopy on 10 mg of
lyophilized Arabidopsis plants treated with nitric acid at 80°C
overnight and diluted one-third with water.
RNA Isolation and Gel-Blot Analyses
Total RNA was isolated from Arabidopsis plants (ecotype Col) as
described by Prescott and Martin (1987). Total RNA was isolated from
the fifth and sixth leaves (excluding cotyledons) of Arabidopsis (ecotype Ler). Total RNA was purified from leaf samples using an RNA
isolator (Genosys Biotechnologies, The Woodlands, TX) following the
manufacturer's instructions. Total RNA was quantified by
spectrophotometry and by 18S rRNA abundance as visualized by ethidium
bromide staining on agarose gels and by hybridization to a radiolabeled
18S rRNA probe. For RNA blots, equal amounts of RNA were separated by
denaturing-agarose gel electrophoresis and transferred to a nylon
membrane as described previously (Sambrook et al., 1989). RNA blots
were probed with [32P]ATP-labeled cDNA probes
and analyzed using a phosphor imager (Molecular Dynamics, Sunnyvale,
CA), a radioanalytical imaging system (InstantImager 2024, Packard,
Canberra, Australia), or by exposure to x-ray film at 80°C with an
intensifying screen.
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RESULTS |
Identification of the CCH Gene
We have previously isolated a gene, SAM45, which is
up-regulated at the mRNA level in soybean cell cultures that are
cytokinin depleted (Crowell and Amasino, 1991). Cytokinins are known to prevent leaf senescence, and removal of cytokinin from cell cultures causes some symptoms of senescence. We identified an Arabidopsis homolog of SAM45 in the expressed sequence tag collection
(accession no. 21536) (Newman et al., 1994) with 97% identity to SAM45
at the amino acid level. The 585-bp cDNA clone contained an open reading frame capable of encoding a 121-amino acid polypeptide with a
molecular mass of 13 kD and a pI of 4.9 (Fig.
1A). The reading frame shown was the only
possible open reading frame in the cDNA and had both a start and stop
codon, indicating that the sequence contained the complete coding
region. Similar sequences were identified from S. cerevisiae
(ATX1; Lin and Culotta, 1995) and from rice (OsATX1;
accession no. DBEST 70798). ATX1 is involved in copper
trafficking (Lin et al., 1997), and because the Arabidopsis gene
corresponding to cDNA 31B12T7 has a similar function (see below), we
refer to it as Copper Chaperone. DNA-blot analysis indicated
that there was a single copy of CCH in the Arabidopsis genome (data not shown).
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| Figure 1.
CCH sequence analysis. A, Nucleotide and predicted
amino acid sequences for the CCH cDNA. B, Alignment of
yeast ATX1 with the predicted amino acid sequence of HAH1 (human),
ATOX1 (mouse; accession no. AF004591), and the 69 N-terminal amino
acids of CCH (Arabidopsis), SAM45 (soybean), and rice ATX1 (OsATX1;
accession no. DBEST 70798). Conserved residues in the alignment are
shaded. The putative metal-binding domain is boxed. C, Amino acid
sequence features of the C-terminal region of CCH. Charged residues are
shown in boldface and their charges are indicated below them.
Secondary-structure prediction suggested that an  -helix could be
formed in the shaded region.
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The alignment of ATX1 with the predicted sequence of CCH and other
similar amino acid sequences is shown in Figure 1B. The amino acid
sequence of CCH and ATX1 are 36% identical and 54% similar across the
length of ATX1. CCH exhibits the same degree of similarity to ATX1
homologs from mouse (ATOX1) and human (HAH1). CCH is 67% identical to
a potential ATX1 homolog from rice (OsATX1). Both the CCH and
OsATX1 polypeptides conserve the motif GMXCXXC (boxed in Fig. 1B),
which is also described at the N-terminal region of copper-pumping
ATPases such as CopA from Enterococcus hirae and
the human Menkes and Wilson disease gene products (Solioz et al.,
1994). This sequence is similar to the more general heavy-metal-binding motif (MTCXXC) found in ATX1 and other metal-binding proteins (Lin and
Culotta, 1995). This motif has been shown to bind copper (Pufahl et
al., 1997).
CCH extended 48 amino acids beyond the C-terminal end of ATX1; this
domain contained 44% charged amino acids, many of which were separated
by a single nonpolar amino acid (Fig. 1C). The alternating
opposite-charged amino acids observed within the CCH C-terminal region
suggests that an -helix could be formed from residues 83 to 100, with a spatial distribution of basic amino acids on one side and acidic
amino acids on the other. Thus, CCH may be composed of two different
domains, an N-terminal region involved in copper binding and the highly
charged C-terminal region, which may be involved in interactions with
other molecules.
Complementation of atx1 and sod1
Mutant Yeast Strains
ATX1 is involved in the transport of copper ions to the secretory
pathway, where the copper is made available to a copper-dependent oxidase essential for high-affinity iron uptake at the plasma membrane
(Pufahl et al., 1997; Valentine and Gralla, 1997). Thus, the
atx1 mutant strain is deficient in high-affinity iron
uptake. In the presence of the iron chelator ferrozine, yeast cells
acquire iron through the high-affinity uptake pathway. Thus,
atx1 mutants cannot grow on ferrozine-containing medium
unless supplemented with excess iron (Lin et al., 1997). To determine
if CCH could replace the function of ATX1 in high-affinity iron uptake,
CCH was expressed in an atx1 mutant strain. The
complete open reading frame of CCH was inserted into a
multicopy vector, allowing constitutive expression of CCH in
yeast. Transformants were plated on medium with and without
supplemental iron (Fig. 2). Strains
expressing either ATX1 or CCH grew in the absence
of supplemental iron, demonstrating that high-affinity iron uptake was
restored by expression of either ATX1 or CCH.
Although restored growth on ferrozine was slow, the growth rate was
comparable to that of the wild type (data not shown).
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| Figure 2.
CCH restored high-affinity iron uptake to
atx1 mutant yeast. The atx1 mutant strain
SL215 was transformed with vector p413-A1 (ATX1
expression), pRS413 (expression vector, negative control), pB-038
(CCH expression), or pSM703 (expression vector, negative
control) and streaked on plates in the positions indicated. All plates
contained ferrozine, an iron chelator. Plates contained synthetic
dextrose medium plus 3 mM ferrozine (SD+fer) or synthetic
dextrose medium plus 3 mM ferrozine plus 50 µM ferrous ammonium sulfate (SD+fer+Fe). Plates were
photographed after incubation at 30°C for 4 d.
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ATX1 was identified by its ability to protect against oxygen toxicity
when expressed in a yeast strain lacking the SOD genes SOD1
and SOD2 (Lin and Culotta, 1995). Phenotypes of
SOD1 deficiency in aerobically grown yeast include retarded
growth rate and auxotrophy for Lys and Met (Liu et al., 1992). The
synthetic pathways for these amino acids contain steps that are
hypersensitive to reactive oxygen (Liu et al., 1992; Slekar et al.,
1996). In the sod1 mutant, ATX1 expressed from a
multicopy expression vector can restore Lys and Met biosynthesis,
allowing the transformed strain to grow without supplemental Lys or Met
(Lin and Culotta, 1995).
To determine whether CCH could restore Lys and Met synthesis to
aerobically grown, SOD-deficient yeast, we expressed CCH in the mutant strain (Fig. 3). The
untransformed SOD-deficient yeast could grow on complete medium but
failed to grow in the absence of Lys or Met. The same strain expressing
either ATX1 or CCH could grow with Lys and Met absent from the medium,
indicating that CCH expression in aerobically grown
SOD-deficient yeast protects the cells from oxygen toxicity to the
extent that Lys and Met synthesis are restored. Thus, CCH function can
replace ATX1 function in this strain.
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| Figure 3.
CCH can restore Lys and Met synthesis in
aerobically grown sod1 mutant yeast. Strain KS107
(sod1) was plated after transformation with no
expression vector (A, negative control), pRS-A1 (B, ATX1
expression), or pB-038 (C, CCH expression). Plates
contained complete medium (SD) or medium lacking Met
(met) or Lys (lys). Plates were photographed after aerobic growth
at 30°C for 2 d.
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To determine whether CCH protects sod1 yeast in a
copper-dependent manner, a sod1 strain transformed with the
CCH expression construct was grown in the presence of BCS, a
copper chelator (Fig. 4A). BCS chelated
copper in the growth medium, making it unavailable to the growing
cells. In the absence of BCS, CCH expression allowed
sod1 yeast to grow in aerobic conditions at a rate similar to that of sod1 yeast grown in anaerobic conditions. When
free copper was chelated by BCS, the growth of the
CCH-expressing sod1 cells in aerobic conditions
was inhibited and was similar to that of sod1 cells
transformed with the vector only. When excess
CuSO4 was added to medium containing BCS, copper
became available to the cells; under these conditions, growth was
restored to CCH-expressing sod1 yeast in aerobic
conditions. As additional evidence for the requirement of copper in CCH
function, CCH was expressed in
sod1,ctr1 double mutants. In these mutants the
absence of CTR1, a copper transporter at the plasma
membrane, prevents uptake of copper to the cytoplasm (Dancis et al.,
1994). CCH expression was unable to restore Lys synthesis in
sod1,ctr1 double mutants (Fig. 4B). These
results indicate that the ability of CCH to protect
sod1 yeast from reactive oxygen toxicity is
dependent on copper availability.
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| Figure 4.
CCH rescued sod1 mutant yeast in a
copper-dependent manner. A, Strain KS107 (sod1) was
transformed with the vector pSM703 (+vector) as a negative control or
pB-038 (+CCH) as the CCH expression
vector. Yeast was grown in yeast extract-peptone-dextrose medium
alone or yeast extract-peptone-dextrose plus 150 µM BCS
with or without 50 µM CuSO4.
A600 was determined after yeast was grown in
either aerobic or anaerobic conditions for 18 h beginning at
A600 = 0.01. B, A, SL201
(sod1,ctr1) plus pSM703 (empty
expression vector); B, KS107 (sod1) plus pSM703; C,
SL201 plus pB-038 (CCH expression); and D,
KS107 plus pB-038 yeast were grown in aerobic or anaerobic conditions
for 2 d on synthetic dextrose medium lacking Lys.
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CCH Gene Expression
RNA-blot analysis established that CCH mRNA was
expressed in the root, stem, leaf, inflorescence, and silique (not shown). To determine the expression of CCH during leaf
senescence, total RNA was isolated from the fifth and sixth leaves of
Arabidopsis at 16, 23, and 28 DAG (Fig.
5). In this population, leaves at 16 DAG
were not fully expanded, leaves at 23 DAG were fully expanded but
showed no yellowing, and leaves at 28 DAG were, by visual estimation,
50% yellow and clearly undergoing senescence. In these experiments,
CCH mRNA levels increased by more than 7-fold after the
onset of leaf senescence. The senescence-associated gene
SAG12, which is expressed in a highly senescence-specific
manner, and the chlorophyll a/b-binding protein
CAB, which is rapidly down-regulated during senescence, serve as
positive and negative controls for leaf senescence (Lohman et al.,
1994).
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| Figure 5.
CCH mRNA was up-regulated during leaf senescence.
The fifth and sixth leaves of Arabidopsis plants (ecotype Ler) were
removed at 16, 23, or 28 DAG, and total RNA was extracted and used for
RNA-blot analysis (leaf photographs are representative of leaves at
each time point). RNA blots were created with equal amounts of each RNA
sample, as determined by spectrophotometry and ethidium bromide
staining. Equal loading was confirmed by detection of 18S rRNA with a
radiolabeled probe. RNA blots were probed with
[32P]ATP-labeled cDNA probes for 18S rRNA (control for
equal loading on the RNA blot), CCH cDNA, chlorophyll
a/b-binding protein (CAB), and the
senescence-associated gene (SAG 12).
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Response to Ozone
The expression of ATX1 is induced by oxidative
stress (Lin and Culotta, 1995). To determine whether CCH was
similarly induced, 3- to 4-week-old Arabidopsis plants were treated
with high ozone concentrations (800 nL/L) for 30 or 60 min and total
RNA from the aerial tissues was analyzed for CCH expression
by RNA-blot analysis (Fig. 6). After 30 min of treatment a more than 30% increase in CCH mRNA was
observed. Longer exposures did not increase the level of CCH
transcripts. This modest induction of the CCH message by
ozone treatment was observed in three separate trials.
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| Figure 6.
CCH mRNA levels increased after treatment with
ozone. Arabidopsis plants (3 to 4 weeks old) were subjected to 800 nL/L
ozone treatment for 0, 30, or 60 min in ozone fumigation chambers. Ten
micrograms of total RNA was used for RNA-blot analysis and probed with
either CCH cDNA or 18S rRNA.
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Response to Copper Treatment
To determine whether CCH is regulated by copper, the
fifth and sixth leaves of Arabidopsis plants at 16 DAG were removed and incubated in liquid medium with or without 50 µM
CuSO4 for 18 h. Total RNA isolated from
these leaves was used to generate RNA blots (Fig.
7A). CCH mRNA levels decreased
by more than 5-fold during this treatment, suggesting that
CCH is down-regulated at the mRNA level by high levels of
exogenous copper. Separately, as a control for copper treatment, mRNA
levels of MT1 increased in copper-treated leaves, as has
been observed previously (Zhou and Goldsbrough, 1994). These results
indicate that the CCH message is regulated differently than
the message of a copper-binding metallothionein, at least when
nonphysiologically high levels of copper are applied.
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| Figure 7.
CCH mRNA was down-regulated in response to copper
treatment. A, The fifth and sixth leaves were removed from Arabidopsis
plants (ecotype Ler) at 16 DAG. Leaves were incubated in light for
18 h with gentle shaking in either Murashige and Skoog medium ()
or Murashige and Skoog medium plus 50 µM
CuSO4 (+). Total mRNA was extracted from leaves and
used for RNA-blot analysis. Blots were probed with either 18S rRNA
(control for equal loading on RNA blot), CCH, or
MT1 cDNA. B, Arabidopsis plants (ecotype Col) were
removed from soil and their roots submerged in 1 mM
CuSO4. Kinetics of copper uptake to the aboveground parts
of the plant were measured by atomic absorption at various time points.
C, Total RNA was isolated from plants after the treatment described
above, as well as from plants treated in parallel with water. RNA blots
made from these samples were probed with [32P]ATP-labeled
CCH (see inset). The graph plots the change in CCH mRNA
levels (expressed in cpm on the RNA blot probed with the
CCH probe) over time of treatment. The 0-h time point
was set to 100% and subsequent values were normalized to the 0-h
value.
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To determine if copper could down-regulate CCH
expression in intact plants, 3- to 4-week-old Arabidopsis plants were
removed from soil and their roots were submerged in 1 mM
CuSO4 solutions for different times. Copper
transport into the shoot was followed by atomic absorption of nitric
acid-digested samples (Fig. 7B). Associated with the accumulation of
copper was a decrease in CCH mRNA (Fig. 7C). After 30 min,
RNA blotting revealed that mRNA levels of CCH decreased by
50%. Longer copper treatment further lowered CCH levels.
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DISCUSSION |
We describe the characterization of CCH from
Arabidopsis. CCH encodes a predicted protein of 121 amino acids that,
in the N-terminal region, is similar in sequence to ATX1
from yeast and contains the heavy-metal-binding sequence MXCXXC that is
common to metal-binding proteins in bacteria, fungi, animals, and
plants. The predicted amino acid sequence of CCH includes a highly
charged carboxyl domain that could interact with other molecules,
perhaps in a copper-dependent manner. The homologs of ATX1,
HAH1 (human), and ATOX1 (mouse) encode proteins
of similar length to ATX1, i.e. they do not bear a C-terminal extension
(Klomp et al., 1997).
CCH is a functional homolog of ATX1. Both CCH and
ATX1 can restore Met and Lys biosynthesis to a
SOD1-deficient strain, indicating that CCH and
ATX1 share the ability to protect yeast from some effects of
the oxidative damage produced by SOD deficiency. The observation that
ATX1 levels increase in yeast cells challenged with reactive
oxygen intermediates is consistent with ATX1 being involved
in a response to reactive oxygen (Lin and Culotta, 1995).
It will be interesting to determine whether the CCH gene
product has the ability to suppress oxygen toxicity in Arabidopsis mutants lacking SOD in a copper-dependent manner, as has been described
for yeast (Culotta et al., 1995; Lin and Culotta, 1995). Also, in the
wild type, it would be interesting to determine whether CCH
overexpression might protect against the pernicious effects of ozone
common in industrialized areas. Levels of CCH mRNA increase moderately after ozone exposure, yet it remains to be determined whether this up-regulation is part of a defense against oxidative stress. Because little information exists regarding the interactions between copper metabolism and protection from oxidative stress in
plants, the study of CCH could clarify the relationship
between these two processes. The human homolog of CCH, HAH1, has
distinct regions mediating copper delivery and oxidative defense (Hung et al., 1998). CCH shows high homology to HAH in these regions and may
retain the same functions.
In the yeast S. cerevisiae, ATX1 is part of pathway that
links copper transport to iron uptake at the cell surface (Yaun et al.,
1995; Stearman et al., 1996; Lin et al., 1997; Valentine and Gralla,
1997). ATX1 interacts with the membrane-bound copper transporter CCC2
to deliver copper to the interior of a post-Golgi vesicle (Pufahl et
al., 1997). Ultimately, copper becomes incorporated into a complex
capable of reducing iron outside the cell (Stearman et al., 1996). An
extracellular mechanism for iron reduction serves to reduce Fe(III),
the common extracellular form of iron, to Fe(II) to facilitate uptake.
A homologous copper transport pathway has been identified in humans;
defects in this pathway are the cause of Menkes and Wilson disease, the
symptoms of which result from deficiencies in copper loading of
copper-containing proteins such as ceruloplasmin (Yaun et al., 1995;
Klomp et al., 1997).
The discovery that the CCH gene product can replace ATX1 in
the high-affinity iron uptake pathway indicates that CCH can interact with CCC2 to deliver copper to the post-Golgi vesicles of yeast cells.
It remains to be determined whether plants express genes encoding
functions homologous to those of other members of the yeast
high-affinity iron-uptake pathway. Nevertheless, the physiology of iron
uptake in plants is consistent with the existence of such a pathway. In
many plants uptake of iron requires reduction of Fe(III), the common
form of iron in aerated soils, to Fe(II) in the root zone (Marschner,
1995). CCH mRNA is expressed at a basal level in the roots,
stems, flowers, siliques, and leaves of Arabidopsis. It is not known
whether iron reduction is taking place in all of these tissues or, if
so, whether CCH is needed for that reduction.
Several metallothioneins that are up-regulated at the mRNA level during
leaf senescence have been identified from plants (Buchanan-Wollaston, 1994; Weaver et al., 1997). These metallothioneins are believed to
protect the cell by binding free copper ions in the cytoplasm. Their
up-regulation during leaf senescence suggests that a copper detoxification function is important in senescing leaf cells. The
CCH gene product may serve a similar function. However,
CCH is regulated differently than one senescence-induced
metallothionein, MT1, in nonsenescent leaves treated with
exogenous copper; copper treatment sufficient to induce MT1
mRNA expression eliminates CCH expression at the mRNA level.
Thus, if CCH functions as a copper chelator, it does not appear to do
so in all situations in which copper toxicity threatens the leaf cell.
The yeast ATX1 message is also not induced in cells treated
with exogenous copper (S.-J. Lin and V.C. Culotta, unpublished data).
We have observed in Arabidopsis that CCH is up-regulated
during leaf senescence, suggesting a possible role for CCH function in
copper binding or transport during that process. During senescence nutrients are redistributed from senescing tissue, contributing to
growth in other parts of the plant (Noodén and Leopold, 1988). An
indication of the importance of copper in plant metabolism is that many
plants redistribute copper from leaves before abscission, thus avoiding
copper loss to the environment (Mauk and Nooden, 1992; Drossopoulos et
al., 1994, 1996; Hocking, 1994). Metals exiting the leaves during
senescence are likely to do so by way of the phloem. Recent studies of
metal transport in castor bean indicate that copper and iron can move
through phloem chelated to organic molecules, in particular the amino
acid nicotianamine (Schmidke and Stephan, 1995).
As chloroplasts and their constituent proteins are broken down during
senescence, copper is released. As discussed above, the metallothionein
MT1 is up-regulated at the mRNA level during leaf senescence
and may be involved in the sequestration of copper released during
senescence. Also, CCH mRNA is up-regulated during senescence
and may be involved in copper sequestration during this process.
Furthermore, CCH, as a functional homolog of
ATX1, may be involved in the delivery of copper to the
secretory system in preparation for phloem loading and transport from
senescing leaves. In Arabidopsis we have observed that copper levels
drop by one-half several days after the onset of senescence (E. Himelblau and R.M. Amasino, unpublished data). It will be
interesting to determine if plants in which CCH expression
is eliminated or attenuated during senescence can continue to transport
copper from senescing tissue. Also, reactive oxygen intermediates
accumulate in leaves undergoing senescence (Noodén and Leopold,
1988). As described above, CCH may encode antioxidant
function, suggesting an additional role for CCH expression
during leaf senescence.
| |
FOOTNOTES |
1
E.H. and R.M.A. were supported by the Consortium
for Plant Biotechnology Research (grant no. DE-FG02-97ER20280), E.H.
was supported by the National Institutes of Health (NIH) Biotechnology Training Program (grant no. 5 T32 GM08349), H.M. and L.P. were supported by the Direccion General de Investigacion Cientifica y
Technica Spain (grant no. PB95-0029-C02-02) and the Conselleria d'
Educació y Ciencia de la Generalitat Valenciana, and S.-J.L. and
V.C.C. were supported by the Johns Hopkins National Institute of
Environmental Health Services Center and NIH (grant no. GM RO1
50016).
2
E.H. and H.M. contributed equally to the
manuscript.
*
Corresponding author; e-mail amasino{at}biochem.wisc.edu; fax
1-608-262-3453.
Received December 11, 1997;
accepted April 30, 1998.
The accession number for the nucleotide sequence reported in this
article is U88711.
| |
ABBREVIATIONS |
Abbreviations:
BCS, bathocuproinedisulfonic acid.
DAG, days
after germination.
SOD, superoxide dismutase.
| |
ACKNOWLEDGMENTS |
We thank the Arabidopsis Biological Resource Center (Columbus,
OH) and the Arabidopsis Expressed Sequence Tag Project (Department of
Energy Plant Research Laboratory, Michigan State University, East
Lansing) for providing cDNA sequences and the 31B12T7 clone. We are
grateful to Dr. Peter Goldsbrough for providing the MT1 cDNA
clone. We thank Joaquín Moreno, Pedro Carrasco, Francisco Estruch, Edgar Spalding, and Betania Quirino for critical reading of
the manuscript.
| |
LITERATURE CITED |
Buchanan-Wollaston V
(1994)
Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus.
Plant Physiol
105:
839-846
[Abstract]
Crowell D,
Amasino RM
(1991)
Induction of specific mRNAs in cultured soybean cells during cytokinin or auxin starvation.
Plant Physiol
95:
711-715
[Abstract/Free Full Text]
Culotta VC,
Joh HD,
Slekar KH,
Strain J
(1995)
A physiological role for Saccharomyces cerevisiae copper/zinc superoxide dismutase in copper buffering.
J Biol Chem
270:
29991-29997
[Abstract/Free Full Text]
Dancis A,
Haile D,
Yaun DS,
Klausner RD
(1994)
The Saccharomyces cerevisiae copper transport protein (Ctr1p).
J Biol Chem
269:
25660-25667
[Abstract/Free Full Text]
Dancis A,
Yaun DS,
Haile D,
Askwith C,
Eide D,
Moehle C,
Kaplan J,
Klausner RD
(1994)
Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport.
Cell
76:
393-402
[CrossRef][ISI][Medline]
Drossopoulos JB,
Bouranis DL,
Bairaktari BD
(1994)
Patterns of mineral nutrient fluctuations in soybean leaves in relation to their position.
J Plant Nutr
17:
1017-1035
[ISI]
Drossopoulos JB,
Kouchaji GG,
Bouranis DL
(1996)
Seasonal dynamics of mineral nutrients by walnut tree reproductive organs.
J Plant Nutr
19:
421-434
[ISI]
Halliwell B,
Gutteridge JMC
(1989)
Free Radicals in Biology and Medicine.
Clarendon Press, Oxford, UK
Hocking PJ
(1994)
Dry-matter production, mineral nutrient concentrations, and nutrient distribution and redistribution in irrigated spring wheat.
J Plant Nutr
17:
1289-1308
[ISI]
Hung IH,
Casareno RLB,
Labesse G,
Mathews FS,
Gitlin JD
(1998)
HAH1 is a copper-binding protein with distinct amino acid residues mediating copper homeostasis and antioxidant defense.
J Biol Chem
273:
1749-1754
[Abstract/Free Full Text]
Kalstrom AR,
Levine RL
(1991)
Copper inhibits the protease from human immunodeficiency virus 1 by both cysteine-dependent and cysteine-independent mechanisms.
Proc Natl Acad Sci USA
88:
5552-5556
[Abstract/Free Full Text]
Klomp LWJ,
Lin SJ,
Yaun DS,
Klausner RD,
Culotta VC,
Gitlin JD
(1997)
Identification and functional expression of HAH1, a novel human gene involved in copper homeostasis.
J Biol Chem
272:
9221-9226
[Abstract/Free Full Text]
Koch K,
Pena M,
Thiele D
(1997)
Copper-binding motifs in catalysis, transport, detoxification and signaling.
Chem Biol
4:
549-560
[CrossRef][ISI][Medline]
Lin S,
Culotta VC
(1995)
The Atx1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity.
Proc Natl Acad Sci USA
92:
3784-3788
[Abstract/Free Full Text]
Lin SJ,
Pufahl RA,
Dancis A,
O'Halloran TV,
Culotta VC
(1997)
A role for the Saccharomyces cerevisiae ATX1 gene in copper trafficking and iron transport.
J Biol Chem
272:
9215-9220
[Abstract/Free Full Text]
Liu XF,
Elashvili I,
Gralla EB,
Valentine JS,
Lapinskas P,
Culotta VC
(1992)
Yeast lacking superoxide dismutase.
J Biol Chem
267:
18298-18302
[Abstract/Free Full Text]
Lohman K,
Gan S,
John M,
Amasino RM
(1994)
Molecular analysis of natural leaf senescence in Arabidopsis thaliana.
Physiol Plant
92:
322-328
[CrossRef]
Marschner H
(1995)
Mineral Nutrition of Higher Plants.
Academic Press, London
Mauk CS,
Nooden LD
(1992)
Regulation of mineral redistribution in pod-bearing soybean explants.
J Exp Bot
43:
1429-1440
[Abstract/Free Full Text]
Murashige T,
Skoog F
(1962)
A revised medium for rapid growth and bioassay with tobacco tissue culture.
Physiol Plant
15:
473-497
[CrossRef]
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]
Noodén LD,
Leopold AC
(1988)
Senescence and Aging in Plants.
Academic Press, San Diego, CA
Prescott A,
Martin C
(1987)
A rapid method for the quantitative assessment of levels of specific mRNAs in plants.
Plant Mol Biol Rep
4:
219-224
Pufahl R,
Singer C,
Peariso K,
Lin S-J,
Schmidt J,
Fahrni C,
Cizewski Culotta V,
Penner-Hahn J,
O'Halloran T
(1997)
Metal ion chaperone function of the soluble Cu(I) receptor Atx1.
Science
278:
853-856
[Abstract/Free Full Text]
Rose MD,
Winston F,
Hieter P
(1991)
Methods in Yeast Genetics: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Schmidke I,
Stephan UW
(1995)
Transport of metal micronutrients in the phloem of castor bean (Ricinus communis) seedlings.
Physiol Plant
95:
147-153
[CrossRef]
Sikorski RS,
Hieter P
(1989)
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics
122:
19-27
[Abstract/Free Full Text]
Slekar KH,
Kosman DJ,
Cizewski Culotta V
(1996)
The yeast copper/zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection.
J Biol Chem
271:
28831-28836
[Abstract/Free Full Text]
Solioz M,
Odermatt A,
Krapf X
(1994)
Copper pumping ATPases: common concepts in bacteria and man.
FEBS Lett
346:
44-47
[CrossRef][Medline]
Stearman R,
Yaun DS,
Yamaguchi-Iwai Y,
Klausner RD,
Dancis A
(1996)
A permease-oxidase complex involved in high-affinity iron uptake in yeast.
Science
271:
1552-1557
[Abstract]
Tamai KT,
Gralla EB,
Ellerby LM,
Valentine JS,
Thiele DJ
(1993)
Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase.
Proc Natl Acad Sci USA
90:
8013-8017
[Abstract/Free Full Text]
Valentine J,
Gralla EB
(1997)
Delivering copper inside yeast and human cells.
Science
278:
817-818
[Free Full Text]
Vulpe CD,
Packman S
(1995)
Cellular copper transport.
Annu Rev Nutr
15:
293-322
[ISI][Medline]
Weaver LM, Himelblau E, Amasino RM (1997) Leaf senescence: gene
expression and regulation. In JK Setlow, ed, Genetic
Engineering, Vol 19. Plenum Press, New York, pp 215-234
Yaun DS,
Stearman R,
Dancis A,
Dunn T,
Beeler T,
Klausner RD
(1995)
The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.
Proc Natl Acad Sci USA
92:
2632-2636
[Abstract/Free Full Text]
Zhou J,
Goldsbrough PB
(1994)
Functional homologs of fungal metallothionein genes from Arabidopsis.
Plant Cell
6:
875-884
[Abstract]
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Top
Abstract
Introduction