Plant, Cell and Environment (2010) 33, 1656–1670
Functional comparison of catalase genes in the
elimination of photorespiratory H2O2 using promoter- and
3⬘-untranslated region exchange experiments in the
Arabidopsis cat2 photorespiratory mutant
YE-QIN HU, SHENG LIU, HONG-MEI YUAN, JING LI, DA-WEI YAN, JIAN-FENG ZHANG & YING-TANG LU
Key Lab of MOE for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China
ABSTRACT
Photorespiration-associated production of H2O2 accounts
for the majority of total H2O2 in leaves of C3 plants and is
mainly eliminated by catalases. In Arabidopsis, lack of
CAT2, but not CAT1 or CAT3, results in growth suppres-
sion and a marked accumulation of H2O2 in leaves. To
evaluate the contribution of individual catalase genes and
their promoters to catalase function, we investigated the
growth suppression and H2O2 accumulation phenotypes of
Arabidopsis derivatives expressing catalase genes from het-
erologous CAT promoters in a cat2 mutant background.
The expression of CAT2 from the CAT2 promoter restored
the wild-type phenotype in a cat2-1 mutant, while CAT1
and CAT3 promoter-driven expression of CAT2 did not.
Ectopic expression of CAT3 from the CAT2 promoter also
restored the normal phenotype, unlike that of CAT1 which
required replacement of the CAT1 3⬘-untranslated region
(UTR) with that of CAT2. These results demonstrated that
the photorespiratory role of CAT2 is determined mainly by
the regulation of its promoter activity. The 3⬘-UTR of CAT2
was vital for controlling CAT2 protein levels under photo-
respiratory conditions. Identification of component of
heterotetramers catalase isoforms suggested that there is
some functional redundancy between CAT2 and CAT1 and
CAT3.
Key-words: 3′-untranslated region; Arabidopsis; catalase;
hydrogen peroxide; leaf; photorespiration; promoter-
exchange experiments.
INTRODUCTION
H2O2 is one of the major reactive oxygen species (ROS),
and plays an important role in plant biological processes
such as seed germination, root elongation (Kwak et al.
2003), auxin-regulated root gravitropism (Joo, Bae &
Lee 2001), stomatal closure (Murata et al. 2001) and
Correspondence: Y. T. Lu. Fax: +86 027 68756380; e-mail: yingtlu@
whu.edu.cn
1656
doi: 10.1111/j.1365-3040.2010.02171.
pce_2171 1656..1670
biotic/abiotic stress responses (Dat et al. 2000; Apel & Hirt
2004). Photorespiratory H2O2, which is generated during
photorespiration in peroxisomes, is the primary source of
H2O2 in C3 plants (Noctor et al. 2002; Karpinski et al. 2003).
Although antioxidative enzymes such as superoxide dismu-
tase (SOD) and ascorbate peroxidases (APX) are active
in leaf peroxisomes, catalase is the foremost scavenging
enzyme for the degradation of photorespiratory H2O2
(Kendall et al. 1983;Willekens et al. 1997; Corpas, Barroso &
del Río 2001; Vandenabeele et al. 2004). Catalase is a tet-
rameric iron porphyrin that catalyses the dismutation of
H2O2 to water and oxygen. While low levels H2O2 are elimi-
nated by APX and other peroxidases with the aid of various
reductants like ascorbate and glutathione, catalase, which
degrades H2O2 without any reducing power, is mainly active
at relatively high H2O2 concentrations (Gechev et al. 2006).
With the exception of CAT-3 from maize, which is mito-
chondrial (Scandalios et al. 1980), all plant catalases studied
to date have been shown to localize to the peroxisome
(Scandalios 1990; Kamada et al. 2003). Thus, catalase plays a
principle role in breaking down H2O2 accumulated in per-
oxisomes (Gillham & Dodge 1986).
There is growing evidence that catalase plays multiple
roles in a variety of plant tissues at different developmental
stages, and it has been implicated in the signal transduction
pathways of various stress responses (Fukamatsu, Yabe &
Hasunuma 2003; Verslues et al. 2007). Mutant and trans-
genic plants with reduced catalase expression exhibit
necrotic lesions, are sensitive to oxidative and salt stress,
and express pathogenesis-related, antioxidant, heat shock
protein and repress anthocyanin biosynthesis genes upon
exposure to high light (Loprasert et al. 1996; Willekens et al.
1997; Vandenabeele et al. 2004; Vanderauwera et al. 2005;
Queval et al. 2007). A recent study of the effect of catalase
deficiency on ion homeostasis revealed a cross-talk between
oxidative stress and ethylene (Bueso et al. 2007).
Like all higher plants, the Arabidopsis genome contains
three catalase genes (At1g20630, At4g35090 and
At1g20620), which encode three individual subunits that
associate to form at least six catalase isoforms (Frugoli et al.
1996). Although the nucleotide and corresponding amino
© 2010 Blackwell Publishing Ltd

acid sequences of the three genes are highly conserved
(Frugoli et al. 1996; McClung 1997), their spatial and tem-
poral expression patterns are quite different. For example,
all three genes are highly expressed in inflorescences, but
only CAT2 and CAT3 are highly expressed in leaves under
the control of a circadian clock (Frugoli et al. 1996; Zhong
et al. 1997). Further analysis of promoter::b-glucuronidase
(GUS) fusions in transgenic plants showed that CAT2 and
CAT3 are expressed in different areas in leaf tissues (Zim-
mermann et al. 2006). CAT2 expression is observed in pho-
tosynthetically active tissues and is down-regulated during
leaf senescence, whereas CAT3 expression is induced with
age and corresponds to accumulated H2O2 in vascular
bundles. Interestingly, the mRNA abundance of CAT genes
increases in a stress-specific manner (Xing, Jia & Zhang
2007; Du et al. 2008). CAT1 may act as a feedback regulat-
ing of ROS signalling, because its expression and activity
are activated by abiotic stresses (Du et al. 2008). Recent
studies have reported that cat2 mutants exhibit suppressed
growth under normal growth conditions and have a defec-
tive photorespiration phenotype (Bueso et al. 2007; Queval
et al. 2007). Although the expression patterns of catalase
genes have been explored, the contribution of their expres-
sion patterns to catalase function is still unclear. Moreover,
data on the specificity and molecular properties of the three
catalase gene products are scarce.
Here, we identified and characterized T-DNA insertion
mutants of catalase gene family members. Only cat2
mutants exhibited significant growth retardation and dis-
tinct accumulation of H2O2 in leaves. To gain further insight
into the functions of the three catalase genes, we examined
the spatial and temporal expression patterns of the catalase
genes, and the effects of catalase expression from heterolo-
gous CAT promoters on the cat2 mutant phenotype.We also
explored the regulatory function of the 3′-UTR of CAT2
on post-transcriptional regulation of catalase genes. These
results indicated no obvious functional differences among
the three catalase gene products, and that the photorespi-
ratory role of CAT2 is determined mainly through the regu-
lation of promoter activity and the 3′-untranslated region.
MATERIALS AND METHODS
Plant materials and growth conditions
The mutant Arabidopsis lines cat1-1 (CS822253), cat2-1
(salk_076998) and cat3-1 (salk_092911) were obtained from
the Arabidopsis Biological Resource Centre (Columbus,
OH, USA). Plants were sown in vermiculite in pots
under controlled environmental conditions (23 ⫾ 2 °C,
200 mmol m-2 s-1, and a 16 h photoperiod) and given irri-
gated nutrient solution three times per week.
Total RNA extraction and semi-quantitative
RT-PCR
Total RNA was isolated using the TRIzol reagent (Invitro-
gen, Carlsbad, CA, USA) according to the manufacturer’s
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
Characterization of Arabidopsis catalase genes 1657
instructions.After treatment of 1 mg of total RNA with RQ1
RNase-free DNase (Promega, Madison, WI, USA), first-
strand cDNA synthesis was carried out using M-MLV
reverse transcriptase (Promega) according to the manufac-
turer’s instructions.
For semi-quantitative PCR analysis, 0.5 mL of the 20 mL
reverse transcription reaction was subjected to PCR (final
volume of 30 mL) using gene-specific primers.As an internal
control, we analysed ubiquitin, which is constitutively
expressed. Each PCR assay was carried out independently
at least three times. The genes analysed and the correspond-
ing specific primers are listed in Supporting Information
Table S1.
Detection of H2O2 in Arabidopsis leaves
Plants were grown in soil for 3 weeks and leaves were
excised. H2O2 contents were determined by the POD-
coupled assay protocols described by Veljovic-Jovanovic
et al. (2001). About 0.1 g Arabidopsis leaves were ground in
liquid N2 and the powder was extracted in 2 mL 1 M HClO4
in the presence of insoluble polyvinylpyrrolidone (5%).The
homogenate was centrifuged at 12 000 g for 10 min, and the
supernatant was neutralized with 5 M K2CO3 to pH 5.6 in
the presence of 100 mL 0.3 M phosphate buffer, pH 5.6. The
solution was centrifuged at 12 000 g for 1 min, and the
sample was incubated for 10 min with one unit ascorbate
oxidase to oxidize ascorbate prior to assay. The reaction
mixture was composed of 0.1 M phosphate buffer, pH 6.5,
3.3 mM 3-(dimethylamino) benzoic acid, 0.07 mM
3-methyl-2-benzothiazoline hydrazone and 0.3 units peroxi-
dase. The reaction was initiated by addition of 200 mL
sample. The absorbance change at 590 nm was monitored
at 25 °C.
Generation of transgenic plants for
promoter-GUS analysis, promoter- exchange
experiments and 3⬘-UTR-exchange experiments
Promoter and genomic fragments of catalase genes were
amplified from genomic DNA of A. thaliana ecotype Col-0
using the appropriate primers (Supporting Information
Table S1), and error-free subcloned PCR products were
confirmed by sequencing. For promoter analysis, each pro-
moter fragment was cloned into the binary vector pBI101.2,
which carries GUS and a selectable marker gene for kana-
mycin resistance. For promoter-exchange experiments, the
promoter fragments of CAT1 and CAT2 were subcloned
into the BamHI site of the binary vector pCAMBIA1301,
while that of CAT3 was subcloned into the SmaI-PstI sites.
The genomic sequences of CAT1 and CAT3 were cloned
into the HindIII site of the vector containing the CAT2
promoter to generate pCAT2::CAT3 and pCAT2::CAT1,
respectively, and the genomic fragment of CAT2 was cloned
into the BamHI site of the vector containing CAT1 pro-
moter and the PstI site of the vector containing CAT3
promoter to generate pCAT1::CAT2 and pCAT3::CAT2,
1658 Y-Q. Hu et al.
respectively. For the 3′-UTR-exchange experiments, each
catalase genomic fragment and corresponding 3′-UTR was
amplified separately. We then used both genomic fragment
and the 3′-UTR as templates to amplify the corresponding
chimeric gene, as illustrated in Fig. S1. For example, to
construct CAT1–CAT2, the CAT1 genomic fragment and
3′-UTR of CAT2 were used as templates. Chimeric genes
were cloned into pCAMBIA1301, which contained the
CAT2 promoter. The junction sites of all subcloned frag-
ments were confirmed by sequencing. Plasmid constructs
were introduced into Arabidopsis by Agrobacterium tume-
faciens (GV3101)-mediated transformation using the floral
dip method (Clough & Bent 1984). After selection of trans-
formed plants on medium containing 50 mg L-1 kanamycin
or 50 mg L-1 hygromycin, the presence of each transgene
was verified by PCR using a gene-specific forward primer
and a vector-specific reverse primer (see Supporting
Information Table S1).
Antibody production
CAT2 was amplified by PCR and subcloned into the
EcoRI site of pET28a for expression in Escherichia
coli BL21 (DE3). The primers used for PCR were
pET28aCAT2F and pET28aCAT2R (see Supporting
Information Table S1). All procedures for protein expres-
sion and purification were carried out as previously
described (Li, Hua & Lu 2006). The peptide (CLGQKLA-
TRLNVRPNF) specific for CAT1 protein was synthesized
by the Fmoc (9-fluorenylmethyloxycarbonyl) method
(Atherton & Sheppard 1989).
A rabbit (New Zealand white rabbit) polyclonal antise-
rum against recombinant AtCAT2 was prepared by subcu-
taneous administration of four injections of 300 mg of
purified recombinant AtCAT2. For the first injection,
protein was mixed with 0.15 mL of complete Freund’s
adjuvant plus 0.11 mL of incomplete adjuvant. The anti-
peptide antibody was produced from a rabbit by injecting
with the synthetic peptide conjugated to BSA, which
had been activated by MBS (m-maleimidobenzoly-N-
hydroxysuccinimide ester). Subsequent booster injections
were administered at approximately 2 week intervals, and
contained protein mixed (1:1) with incomplete adjuvant.
Blood was collected 2 weeks after the last injection. Anti-
serum was collected following separation from clotted red
blood cells by centrifugation (2500 g for 15 min) and stored
at -80 °C.
Western blot analysis
Tissue extracts containing 20 mg of total protein were mixed
with 2 mL of 5XSDS-PAGE sample buffer (Sambrook et al.
1989) and then heated for 3 min at 95 °C. Proteins were
separated by 10% SDS-polyacrylamide gel electrophoresis
and then electroblotted onto a nitrocellulose membrane.
The membrane was blocked in 5% non-fat dry milk in
TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1%
Tween 20) overnight at 4 °C and then washed three times in
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
TBS-T for 10 min each wash. The membrane was incubated
with anti-AtCAT2 polyclonal antibody (1:500) diluted in
1% BSA in TBS-T for 2 h at room temperature. The mem-
brane was washed in TBS-T three times for 10 min each,
and then incubated for 1 h with goat anti-rabbit IgG (1:5000
dilution in TBS-T) conjugated to horseradish peroxidase.
Following three washes for 10 min each in TBS-T, immu-
noreactive proteins were detected using a DAB substrate
solution.
Gel analysis and activity assays of
catalase isoforms
Tissue from various organs was harvested and frozen at
-80 °C until use. Because CAT2 and CAT3 gene expres-
sion is regulated by circadian rhythm, tissues were always
harvested 6 h after the beginning of illumination. Tissues
were ground to a powder in liquid nitrogen and then
homogenized in a solution of 100 mM Tris-HCl, pH 8.0,
20% glycerol and 30 mM dithiothreitol (DTT) at 4 °C.
After centrifugation at 14 000 g for 30 min at 4 °C, the
supernatant was recovered and protein concentration was
quantified according to the method of Bradford (1976)
using bovine serum albumin (BSA) as the standard.
Protein extracts containing 20 mg of total protein were
separated by 7.5% native polyacrylamide gel electro-
phoresis with a 3.5% stacking gel for 14 h (70 V) at 4 °C in
electrophoresis buffer (250 mM glycine, 25 mM Tris-HCl,
pH 8.3). Subsequently, the gels were stained for catalase
activity according to the method of Clare et al. (1984).
Catalase activity assays were performed according to
Veljovic-Jovanovic et al. (2001).
GUS staining
GUS staining was carried out according to the methods
of Jefferson (1987). Briefly, tissues or whole plants
were incubated at 37 °C overnight in staining solution
(100 mM sodium phosphate buffer, pH 7.5, containing
10.0 mM EDTA, pH 8.0, 0.5 mM K3[Fe(CN)6] and
0.5 mM K4[Fe(CN)6], 0.1% Triton X-100 and 1.0 mM
5-bromo–chloro-3-indolyl-b-D-glucuronide). Chlorophyll
was removed by rinsing the plant material in 70% ethanol.
RESULTS
Genomic characterization of cat1-1, cat2-1,
cat3-1, and cat2-1/cat3-1 insertion mutants
of Arabidopsis
To obtain knockout mutants of the three Arabidopsis cata-
lase genes, we selected T-DNA insertion lines from the
Arabidopsis Biological Resource Center (ABRC) (Fig. 1a).
Using PCR with specific primers to analyse plants grown
from T3 seeds, we identified homozygous T-DNA insertion
lines (Fig. 1b), which we designated cat1-1, cat2-1 and
cat3-1. T4 seeds obtained from the identified homozygotes
were used for all further analyses. Because CAT2 and CAT3
 Characterization of Arabidopsis catalase genes 1659

Figure 1. Genomic organization of cat mutants. (a) Structures of genes CAT1, CAT2 and CAT3 are shown with insertion sites of T-DNA
in knockout plant lines. Exons are indicated by blocks, introns by real lines, untranslated regions by broken lines and primers for the
genetic characterization by small black arrows. (b) Genomic characterization of T-DNA insertion lines. PCR with genomic DNA as
template showed the presence of T-DNA in each catalase gene in the appropriate mutant; lack of the wild-type-specific product showed
that the T-DNA allele was homozygous. Each wild-type (w) and T-DNA (T) primer pair was specific for the gene of the indicated mutant.
(c) T-DNA insertions resulted in loss of mRNA of the three catalase genes as shown by RT-PCR. Amplification of ubiquitin from the
same cDNA preparations was used as a positive control.

are highly expressed in leaves (Frugoli et al. 1996), we gen-
erated crosses of cat2-1 and cat3-1 to obtain a homozygous
double insertion mutant, which was also confirmed by PCR
(Fig. 1b). The resulting double mutant was designated cat2-
1/cat3-1. When we analysed catalase gene expression in
each of the cat mutants using RT-PCR and gene-specific
primers that targeted the 3′-UTRs of the genes, we were
unable to detect corresponding transcripts in any of the
mutants (Fig. 1c), indicating that they were complete gene
knockouts.
approximately 33% that of wild-type fresh weight (Fig. 2c).
The cat2-1/cat3-1 double mutant exhibited a slightly more
severe phenotype than the cat2-1 mutant (Fig 2–c). These
observations were consistent with previous reports by
others (Bueso et al. 2007; Queval et al. 2007). Compared to
wild-type plants, cat1-1, cat2-1, cat3-1, and cat2-1/cat3-1
exhibited variable reductions in catalase activity, retaining
92%, 24%, 83% and 7% of total wild type catalase activity,
respectively (Fig. 2d).

Expression patterns of CAT1, CAT2, and CAT3

Morphological and biochemical
characterization of cat mutants
The cat2 mutant was previously characterized as a
photorespiratory mutant under standard irradiance of
200 mmol m-2 s-1, which is approximately 50% saturating for
photosynthesis in the Columbia ecotype of A. thaliana cul-
tivated in a controlled environment growth chamber
(Queval et al. 2007). Under similar conditions, cat1-1 and
cat3-1 were indistinguishable from wild-type plants (Fig. 2).
By comparison, cat2-1 exhibited an altered morphology,
including pale green colour, up-curled leaves and reduced
size (Fig. 2a). There was a greater accumulation of H2O2 in
the leaves of cat2-1 (Fig. 2b), and plant fresh weight was
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
in leaves
Previous work has shown that deletion of CAT2, but not
CAT1 or CAT3, results in a growth suppression phenotype
and obvious accumulation of H2O2 in leaves. To determine
the mechanism of this differential effect of catalase gene
expression on the elimination of photorespiratory H2O2,
we generated transgenic plants containing various CAT
promoter::GUS fusion constructs, and examined the expres-
sion pattern of each construct in leaves. Previously, the
expression pattern of catalase genes in different tissues
was analysed during senescence using a similar approach
(Zimmermann et al. 2006). The effect of catalase gene
expression on plant growth was also studied. To gain further
1660 Y-Q. Hu et al.

Figure 2. Morphological and biochemical characterization of wild-type and cat mutants (a) Top view of the 3-week-old wild-type and cat
mutants. Scale bars represent 1 cm. (b) H2O2 content in leaves of 3-week-old wild-type and cat mutants. Means and SE were calculated
from three independent experiments. FW, fresh weight. (c) Rosette fresh weight of 3-week-old wild-type and cat mutants. Means and SE
were calculated from five plants of each line. (d) Measurements of catalase activity of leaves of 3-week-old wild-type and cat mutants.
Means and SE were calculated from three independent experiments.

insight into the regulation of expression of catalase genes,
we analysed longer promoter sequences, consisting of over
2000 basepairs upstream of the cat start codon. As the
abnormal phenotype of the cat2 mutant predominantly
affected leaves, we focused on GUS activity in the leaves of
transgenic plants. The expression of CAT1 was abundant in
the cotyledon of early seedlings (Fig. 3a), and decreased
with development (Fig. 3b). CAT1 expression was nearly
undetectable in young or mature rosette leaves (Fig 3c & e),
but was induced and began to increase upon senescence
(Fig. 3d). The expression of CAT2 localized predominantly
to the mesophyll cells of green leaves (Fig 3f–h & j). As
yellow cotyledons turned green, CAT2 expression levels
gradually rose (Fig 3f & g). CAT2 expression was down-
regulated during leaf senescence and was much less abun-
dant in senescent leaves (Fig. 3i). CAT3 was highly
expressed in the vasculature of senescent cotyledon and
rosette leaves, but less so in mesophyll cells adjacent to the
vasculature (Fig 3l, m & o). CAT3 was weakly expressed in
senescent leaves (Fig. 3n).
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
Expression of catalase genes under the control
of heterologous CAT promoters differentially
affects the phenotype the cat2-1 mutant
The expression patterns of the three catalase genes in
leaves are quite different. To examine the role of differen-
tial catalase gene expression on catalase function, we placed
expression of the catalase genes under the control of het-
erologous CAT promoters, and examined the effect on the
cat2-1 mutant phenotype. Expression of a genomic frag-
ment incorporating the CAT2 locus under the control of the
CAT2 promoter was able to complement the cat2-1 mutant
phenotype; all 45 cat2-1/pCAT2::CAT2 transgenic plants
were restored to the wild-type phenotype (Fig 4a & c–e).
We then generated the following chimeric genes by placing
catalase coding regions, including part of the 3′-downstream
region, under the control of a heterologous CAT promoter,
resulting in pCAT1::CAT2, pCAT3::CAT2, pCAT2::CAT3
and pCAT2::CAT1. The chimeric genes were introduced
together with the hygromycin resistance marker into a
 Characterization of Arabidopsis catalase genes 1661

(a) (b) (e)

(c) (d)

(f) (g) (j)

(h) (i)

(k) (l) (o)

(m) (n)

Figure 3. Histochemical analysis of expression patterns of Arabidopsis catalase genes. Glucuronidase (GUS) activity in transgenic
Arabidopsis carrying the CAT1 (a–e), CAT2 (f–j), and CAT3 (k–o) promoter::GUS transgenes was analysed by staining with X-Gluc.
Images are shown of young cotyledon (a, f, k) from 3-day-old seedlings, old cotyledon (b, g, l) from 14-day-old seedlings, mature leaves
(c, h, m) and whole plants from 14-day-old plants (e, j, o). Senescence leaves (d, i, n) from 7-week-old plants are also shown. Scale bars
represent 0.5 mm.

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
1662 Y-Q. Hu et al.

Figure 4. Effects of the expression of catalase genes under the control of different CAT promoters on the phenotype of cat2-1 plants.
(a) Top view of the 3-week-old wild-type, cat2-1 and mutant derivatives. Scale bars represent 1 cm. (b) RT-PCR analysis of catalase genes
transcripts in the wild-type, cat2-1 and mutant derivatives. Total RNA was prepared from leaves of 3-week-old plants. Amplification of
ubiquitin from the same cDNA preparations was used as a positive control. The experiment was repeated three times with similar results.
(c) H2O2 content in leaves of 3-week-old wild-type, cat2-1 and mutant derivatives. Means and SE were calculated from three independent
experiments. FW, fresh weight. (d) Rosette fresh weight of 3-week-old wild-type, cat2-1 and mutant derivatives. Means and SE were
calculated from five plants of each line. (e) Measurements of catalase activity of leaves of 3-week-old wild-type, cat2-1 and mutant
derivatives. Means and SE were calculated from three independent experiments.

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
 Characterization of Arabidopsis catalase genes 1663

cat2-1 mutant background. Resultant T1 seeds were col-

lected and sown on MS agar plates containing hygromycin
to identify T1 transformants, and then hygromycin-resistant
T1 plants were transferred to soil and grown to maturity.
After 3 weeks, plants of all the mutant derivatives were
indistinguishable from cat2-1 mutants, with the exception of
cat2-1/pCAT2::CAT3 transformants, which expressed CAT3
under the control of the CAT2 promoter. Among the 49 T1
cat2-1/pCAT2::CAT3 plants, 37 exhibited obvious comple-
mentation, in that leaves were green and a wild-type pattern
of venation was restored. T2 seeds from all of the mutant
derivatives, which were 100% hygromycin-resistant,
were used as the homozygous lines for all subsequent
experiments.
We analysed the leaves of the transgenic lines by
RT-PCR to determine whether the catalase genes were
expressed under the control of different CAT promoters
(Fig. 4b). Transcript levels of CAT1 and CAT3 were
markedly increased in cat2-1/pCAT2::CAT1 and cat2-1/
pCAT2::CAT3, respectively. We also detected the transcrip-
tion of CAT2 in cat2-1/pCAT1::CAT2 and cat2-1/
pCAT3::CAT2, the latter of which had wild-type levels of
transcription of CAT2. Figure 4c shows the accumulation of
H2O2 in the leaves of the different lines. The accumulation
of H2O2 in cat2-1/pCAT2::CAT2 was similar to wild-type.
The H2O2 levels in cat2-1/pCAT2::CAT3 transgenic lines
were significantly reduced as compared to that of the cat2-1
mutant, while in the other mutant derivatives, there were
no obvious differences as compared to the cat2-1 mutant. Figure 5. Characterization of CAT subunits by Western blot
We also examined the fresh weight of rosette leaves in analysis using antibodies against AtCAT2 and AtCAT1.
the different lines (Fig. 4d). The fresh weight of cat2-1/ Coomassie blue staining of the Rubisco protein was used as the
pCAT2::CAT2 leaves was similar to the wild-type, while that loading control. (a) Western blot analyses of SDS-PAGE of
of cat2-1/pCAT2::CAT3 was approximately 80% of the protein extracts (20 mg per lane) from flowers of wild-type and
wild-type. There were no obvious differences in rosette leaf cat mutants. (b) Western blot analyses of SDS-PAGE of protein
extracts (10 mg per lane) from leaves of 3-week-old wild-type,
fresh weights of the other lines as compared to the cat2-1
cat2-1 and mutant derivatives.
mutant. Catalase activity in cat2-1/pCAT2::CAT2 transgenic
plants was similar to wild-type, and there were obvious
increases in catalase activities in cat2-1/pCAT3::CAT2 and highly expressed in this part of the plant (Frugoli et al. 1996;
cat2-1/pCAT2::CAT3 plants (approximately 47% and 66% McClung 1997; Zimmermann et al. 2006). We detected a
of wild type, respectively) (Fig. 4e). The levels of catalase 57 kDa and 60 kDa catalase subunit by immunoblot
activity in cat2-1/pCAT1::CAT2 and cat2-1/pCAT2::CAT1 (Fig. 5a). The 60 kDa subunit was detected in wild-type,
plants did not vary markedly from that of the cat2-1 mutant. cat1-1 and cat2-1 mutant plants, but was absent in cat3-1 and
cat2-1/cat3-1, indicating that this band represented the
Biochemical analysis of catalase protein CAT3 subunit. The 57 kDa catalase subunit was strongly
detected in wild-type, cat1-1 and cat3-1, but was not
function in transgenic lines using
detected in cat2-1 and cat2-1/cat3-1, indicating that it repre-
promoter-exchange experiments
sented the CAT2 subunit.Although the deduced amino acid
While ectopic CAT transcription could be detected in all sequences of CAT1 and CAT2 are 86% identical and 95%
transgenic derivatives, there was no obvious increase similar (Tatusova & Madden 1999), we were unable to
of catalase activity in cat2-1/pCAT1::CAT2 and cat2-1/ detect the CAT1 subunit using our anti-AtCAT2 antibody.
pCAT2::CAT1 plants. To investigate this phenomenon in We then synthesized a CAT1-specific peptide, and used this
more detail, we examined changes of the level of catalase peptide as an antigen to generate a CAT1-specific peptide
subunits in all transgenic derivatives. Full-length recombi- antibody (as described in Materials and Methods). Immu-
nant Arabidopsis CAT2 was expressed and purified using E. noblot analysis using the CAT1-specific peptide antibody
coli, and then the purified protein was used to generate a revealed a 60 kDa catalase subunit, which was absent in
polyclonal anti-AtCAT2 antiserum. We examined catalase cat3-1 and cat2-1/cat3-1, by which we inferred that this band
subunit expression in flowers of Arabidopsis by Western represents the CAT3 subunit, and a 57 kDa catalase subunit
blot analysis, since all three catalase genes are known to be that was absent only from cat1-1, indicating that it
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
1664 Y-Q. Hu et al.

represents the CAT1 subunit. Using these two antibodies,

we examined the levels of catalase subunits in the leaves of
3-week-old transgenic derivatives (Fig. 5b). Compared to
the cat2-1 mutant, the amount of CAT3 in cat2-1/
pCAT2::CAT3 transgenic plants was obviously increased.
The level of CAT2 subunit in cat2-1/pCAT1::CAT2 and
cat2-1/pCAT3::CAT2 derivatives was increased, with the
latter exhibiting a marked increase as compared to the
former, in agreement with the results of RT-PCR analysis.
We were unable to detect increased CAT1 in cat2-1/
pCAT2::CAT1 transgenic plants.
We next carried out a detailed biochemical analysis of
changes in the activities of the different catalase isoforms.
To date, six catalase isoforms have been identified in Ara-
bidopsis by non-denaturing gel electrophoresis (Frugoli
et al. 1996). Based on 3-AT inhibition experiments, three of
the Arabidopsis isoforms have been shown to correspond to
CAT1, CAT2 and CAT3 (Orendi et al. 2001); however, the
exact composition of the isoforms remains unknown. When
we analysed the composition of the catalase tetramer in
wild-type Arabidopsis and cat mutants, we identified seven
different isoforms (Fig. 6a). Isoform one was absent from
the cat1-1 mutant, suggesting that it was composed of only
CAT1. Likewise, we determined that isoform two was com-
posed of only CAT3, while isoform seven was composed of
only CAT2. Isoform three, which was very similar to
isoform two, was a novel isoform. It was absent in cat1-1,
cat2-1 and cat2-1/cat3-1 mutants, indicating that it was com-
posed of CAT1 and CAT2. Similarly, isoform four and
isoform six appeared to be composed of CAT1 and CAT2,
while isoform five was composed of CAT2 and CAT3.
We examined the activity of catalase isoforms in the
leaves of 3-week-old mutant derivatives that were develop-
mentally indistinguishable from wild-type plants. Wild-type
and cat2-1 mutant plants were used as controls (Fig. 6b). We
were unable to detect CAT2 catalase activity in cat2-1/
pCAT1::CAT2 transgenics. There was no obvious change in
the activity of isoform one, which was composed of CAT1,
in cat2-1/pCAT2::CAT1 transgenic plants as compared to
the cat2-1 mutant. In contrast, the activity of isoform two,
which was composed of CAT3, was increased by approxi-
mately 80% in cat2-1/pCAT2::CAT3 transgenic plants, Figure 6. Characterization of catalase isoforms by zymograms
and CAT2 catalase activity was detected in cat2-1/ analysis. (a) Zymogram of catalase isoforms activity in flowers of
pCAT3::CAT2 plants. We then examined catalase activity in wild-type and cat mutants. Horizontal lines indicate the seven
senescent leaves, in which there is a 50–75% loss of chloro- catalase isoforms resolved; (b) Zymogram of catalase activity in
phyll, in 7 week-old plants (Fig. 6c).The activities of isoform leaves of 3-week-old wild-type, cat2-1 and mutant derivatives.
three through six were detected in cat2-1/pCAT1::CAT2, (c) Zymogram of catalase activity in senescence leaves (50–75%
loss of chlorophyll) of 7-week-old wild-type, cat2-1 and mutant
whereas there was still no detectable change in the activity
derivatives. All the experiments were repeated at least four times
of isoform one in cat2-1/pCAT2::CAT1 as compared to the showing always the same results.
cat2-1 mutant.

for the post-transcriptional block in CAT1 expression from

Role of the 3⬘-UTR in the function of CAT2
the CAT2 promoter, we generated CAT1-CAT2 and CAT2-
Although the amino acid sequences of the three catalase CAT1 gene chimeras by exchanging the 3′-untranslated
subunits are 75–84% identical and 87–94% similar (Frugoli regions of CAT1 and CAT2 with each other, and then exam-
et al. 1996), the nucleotide sequences of the 3′-UTRs are ining the expression of the chimeras under the control of
highly divergent among the three cDNAs. To investigate the CAT2 promoter in the cat2-1 mutant background.
whether differences in 3′-UTR sequences were responsible Among the 44 T1 cat2-1/pCAT2::CAT1-CAT2 transgenic
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670

plants, which expressed a chimeric CAT1 coding region and
CAT2 3′-UTR, 39 exhibited full complementation, in that
the levels of H2O2 in leaves and rosette fresh weight were
similar to wild-type plants (Fig 7b & c), and catalase activity
in leaves was restored to 88% of wild-type levels (Fig. 7d).
By comparison, there was a much higher accumulation of
H2O2 in leaves of cat2-1/pCAT2::CAT2-CAT1 transgenic
plants as compared to cat2-1/pCAT2::CAT2 plants (Fig. 7b),
and the rosette fresh weight and catalase activity in leaves
reverted to 50% and 48% of wild type, respectively (Fig 7c
& d). Biochemical analysis of catalase proteins showed that
CAT1 catalase activity was increased in leaves of cat2-1/
pCAT2::CAT1-CAT2 plants, whereas catalase activity con-
tributed by CAT2 in leaves of cat2-1/pCAT2::CAT2-CAT1
plants was decreased significantly as compared to cat2-1/
pCAT2::CAT2 transgenics (Fig 8a & b).
DISCUSSION
Based on molecular analyses of catalase gene expression in
different plants (Ni & Trelease 1991; Willekens et al. 1994;
Suzuki et al. 1995), plant catalases have been subdivided
into three classes (Willekens, Inzé & Van Montagu 1995).
Unlike Class III catalases, which are highly expressed in
young seedlings and senescent tissues, Class I and Class II
catalases are abundant in mature plants. While Class II
catalases are mainly expressed in vascular tissues, Class I
catalases are light-dependent and are most prominent in
photosynthetic cells. Thus, it has been postulated that Class
I catalases are more important in the removal of H2O2
produced during photorespiration than members of the
other classes (Willekens et al. 1994, 1995; Zimmermann
et al. 2006). Recent studies of an Arabidopsis cat2 mutant
have verified this hypothesis (Bueso et al. 2007; Queval et al.
2007). The results of the current study confirm and expand
these earlier findings. Firstly, a major photorespiratory role
of CAT2 was demonstrated by the finding that the loss of
neither CAT1 nor CAT3 resulted in a photorespiration phe-
notype, and the loss of CAT3 did not markedly aggravate
the phenotype of the cat2 mutant (Fig. 2). Secondly, an
important regulatory role of the CAT2 promoter was
demonstrated by complementation experiments, in which
expression of CAT2 and ectopic expression of CAT3 or the
chimeric gene CAT1-CAT2 complemented the cat2 mutant
phenotype when they were placed under the control of the
CAT2 promoter (Figs 4 and 7). Our results suggest that the
mechanism by which CAT1 and CAT3 are restricted from
participating in the photorespiratory process involves regu-
lation at the level of expression. Promoter-GUS analysis
revealed that CAT1, a Class III catalase, is expressed mainly
in senescent leaves (Fig. 3). In agreement with this, in cat2-
1/pCAT1::CAT2 transgenic plants, which expressed CAT2
under the control of the CAT1 promoter, CAT2 isoform
activity in CAT1-expressing regions was detected only in
senescent leaves (50–75% loss of chlorophyll) (Fig. 6c), in
which degradation of chlorophylls and Rubisco leads to a
rapid decline in photosynthetic activity and photorespira-
tion (Friedrich & Huffaker 1980; Jiang et al. 1993), and
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
Characterization of Arabidopsis catalase genes 1665
there were high levels of photorespiratory H2O2 (Fig. 4c).
Although the expression of CAT3 was abundant in mature
leaves, expression was specific to vascular tissues (Fig. 3).
Further analysis revealed that in cat2-1/pCAT3::CAT2
transgenic plants (CAT2 under control of the CAT3 pro-
moter), CAT2-specific isoform activity in CAT3-expressing
regions was readily detected (Fig. 6b). However, while total
catalase activity was increased by approximately 50%
(Fig. 4e), the accumulation of H2O2 in leaves was not mark-
edly decreased (Fig. 4c). In conclusion, the results clearly
demonstrated that the photorespiratory role of CAT2
is determined mainly by the regulation of its promoter
activity
The 3′-UTR is crucial for several aspects of post-
transcriptional regulation, including mRNA transport from
the nucleus into the cytoplasm, enhancement of mRNA
transcript stability and regulation of mRNA translation
(Zhao, Hyman & Moore 1999; Grzybowska, Wilczynska &
Siedlecki 2001; Proudfoot, Furger & Dye 2002). The
absence of a 3′-UTR has been shown to result in low trans-
lational efficiency of cat1 mRNA in rye leaves in vitro
(Schmidt, Grief & Feierabend 2006). In the current study,
we investigated the function of the 3′-UTR of Arabidopsis
CAT2 mRNA in vivo. When driven by the same CAT2
promoter, the replacement of the CAT2 3′-UTR by that of
CAT1 led to obvious decreases in levels of CAT2 subunit as
well as catalase activity contributed by CAT2 (Fig. 8), and
accompanied by a marked increase in the levels of H2O2 in
leaves (Fig. 7b). On the other hand, when driven by the
CAT2 promoter, transcription of CAT1 in leaves increased
significantly, but there were no obvious changes detected in
the levels of CAT1 subunit. When the 3′-UTR of CAT1 was
replaced by that of CAT2, active CAT1 was expressed
under the control of the CAT2 promoter and photorespira-
tory H2O2 was efficiently eliminated (Fig. 8). These results
suggested that the 3′-UTR of CAT2 is vital for regulating
the protein level of CAT2 under photorespiratory
conditions.
There is growing evidence that H2O2 serves as a major
signalling molecule in various aspects of cellular function in
plants (Apel & Hirt 2004; Foyer & Noctor 2005; Vanderau-
wera et al. 2005; Gechev et al. 2006). However, our under-
standing of the mechanisms that control H2O2 production
and metabolism is relatively limited. As a scavenger or
door-keeper for H2O2, catalase plays an important role not
only in protecting cells against oxidant stress, but also in
governing cellular redox homeostasis and by extension, the
regulation of cellular signalling (Baker & Orlandi 1995;
Xing, Jia & Zhang 2008; Foyer et al. 2009). Photorespiration
is by far the fastest H2O2-producing system in photosyn-
thetic cells (Noctor et al. 2002). There is good evidence that
reduced catalase expression under photorespiratory condi-
tions causes dramatic adjustments in the glutathione pool
(Smith et al. 1985; Queval et al. 2007, 2009), a key thiol/
disulfide buffer in cellular redox homeostasis (Foyer &
Noctor 2005). On the other hand, H2O2 production and
signalling under abiotic stresses could also activate the
expression and activity of catalase (Orendi et al. 2001; Xing
1666 Y-Q. Hu et al.

Figure 7. Effects of CAT1-CAT2 and CAT2-CAT1 chimeric gene expression in the CAT2-expression region (a) Top view of the
3-week-old wild-type and cat2-1 and mutant derivatives. Scale bars represent 1 cm. (b) H2O2 content in leaves of 3-week-old wild-type,
cat2-1 and mutant derivatives. Means and SE were calculated from three independent experiments. FW, fresh weight. (c) Rosette fresh
weight of 3-week-old wild-type, cat2-1 and mutant derivatives. Mean values from five plants are shown. (d) Measurements of catalase
activity of leaves of 3-week-old wild-type, cat2-1 and mutant derivatives. Means and SE were calculated from three independent
experiments.

© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670

Figure 8. Biochemical analysis of catalase proteins of
transgenic lines in the 3′-untranslated regions-exchange
experiments (a) Western blot analyses of SDS-PAGE of protein
extracts (10 mg per lane) from leaves of 3-week-old wild-type,
cat2-1 and mutant derivatives using antibodies against AtCAT2
and AtCAT1. Coomassie blue staining of the Rubisco protein
was used as the loading control. (b) Zymogram of catalase
activity in leaves of 3-week-old wild-type, cat2-1 and mutant
derivatives.
et al. 2007; Du et al. 2008). Abscisic acid (ABA) is a well-
known regulator of plant stress tolerance and plant physi-
ology, and ABA-induced H2O2 leads to a clear increase in
the levels of CAT1 transcript and isoform activity in a
process that appears to be mediated by AtMKK1- and
AtMPK6-coupled mitogen-activated protein kinase signal-
ling (Du et al. 2008; Xing et al. 2008). The expression of
CAT3 was enhanced under oxidative stress at the develop-
mental stage, and was accompanied by a clear increase in
CAT3 activity (Orendi et al. 2001). Unlike CAT1 and CAT3,
the enzyme activity of CAT2 does not always correlate with
CAT2 expression (Orendi et al. 2001; Queval et al. 2007),
as reported for Class I catalase in rye (Schmidt et al. 2002).
For example, the expression of CAT2 was enhanced under
oxidative stress while CAT2 activity remained unaffected
(Orendi et al. 2001). A recent study demonstrated that
increasing the rate of photorespiration causes a significant
increase in catalase activity that is accompanied by an
increase in CAT2 expression, but not by an increase in
the CAT2 transcript level (Queval et al. 2007). Hence, a
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
Characterization of Arabidopsis catalase genes 1667
complicated mechanism would appear to adjust the catalase
activity of CAT2 at the post-transcriptional level. The
results of the current study support this hypothesis, and
demonstrate further that the 3′-UTR is a vital element in
the regulation of CAT2 under stress conditions. Taken
together, the results suggest that a mechanism controls the
level of H2O2 in leaves by regulating the level of CAT2
protein, and that this mechanism may depend on the cis-
elements of 3′-UTR of CAT2. When plants are grown under
non-stress conditions (such as low irradiance and high
levels of CO2), CAT2 protein would be maintained at a low
level, resulting in the optimal level of H2O2 for signalling
(Apel & Hirt 2004). Conversely, when plants are transferred
to stress conditions (such as high irradiance and air), the
protein level of CAT2 would increase (Queval et al. 2007).
Thus, the role of CAT2 would be twofold; firstly, it would
protect cells from H2O2 toxicity, and secondly, it would
modulate the level of H2O2 to maintain the optimal redox
state of antioxidants, such as ascorbate or glutathione,
which activate redox signalling pathways (Vanacker, Carver
& Foyer 2000; Michelet et al. 2005; Michelet et al. 2008). Our
study of this 3′-UTR may further facilitate research into
hydrogen peroxide signalling and whole cell redox homeo-
stasis. To avoid interference from other CAT mRNAs, we
used primers based on 3′-UTR sequences to analyse for
mRNA abundance. Unfortunately, these primers were not
suitable for the analysis of the expression of chimeric genes
with exchanged 3′-UTRs. Hence, the precise mechanism of
post-transcriptional regulation of CAT2 by the 3′-UTR
remains to be elucidated.
Unlike animals, which generally have a single catalase
gene, plants contain multiple catalase isoforms that are
encoded by gene families (McClung 1997; Heinze & Ger-
hardt 2002; Feierabend 2005). The roles of catalase isoforms
are still a matter of great interest because the specific func-
tions of some of them have not been fully elucidated
(Feierabend 2005). Catalase isoform arise through oligo-
merization of subunits into tetramers, and higher plants
may simultaneously express homotetrameric and heterotet-
rameric CAT isoforms (Fita & Rossmann 1985; Gouet,
Jouve & Dideberg 1995; Heinze & Gerhardt 2002). When
CAT subunit polypeptides encoded by distinct genes are
simultaneously expressed in the same cell, heterotetramers
of CAT can form (Santos et al. 2006). Identification of the
components of these catalase isoforms will contribute to a
better understanding of their function. A dicotyledonous
plant, Arabidopsis contains three subunits that associate to
form at least six catalase isozymes (Frugoli et al. 1996).
Based on 3-AT inhibition experiments, three of them have
been shown to correspond to CAT1, CAT2 and CAT3
(Orendi et al. 2001). Here, by comparing catalase isoforms
in wild-type and cat mutants, we identified seven different
isozymes (Fig. 6a). Our data showed that three of them
were composed of only CAT1, CAT2, or CAT3 subunits; the
remaining four were composed of CAT2 and either CAT1
or CAT3. These results could reflect the sub-cellular local-
ization of catalase subunits. It is possible that CAT2 local-
izes to the same sub-cellular compartments as CAT1 or
1668 Y-Q. Hu et al.
CAT3, while the localization of CAT1 and CAT3 is distinct.
CAT1, a Class III catalase, is predicted to localize to gly-
oxysomes (Willekens et al. 1995). Glyoxysomes function in
the conversion of lipids into sucrose and are present mainly
in storage organs such as endosperms and cotyledons
during post-germinative growth in oil-seed plants, as well as
in senescent organs (Beevers 1982; Nishimura et al. 1996).
In these tissues, catalase isoforms composed of CAT1 and
CAT2 are readily detected (Zimmermann et al. 2006 and
Fig. 6c). The expression of CAT2 in young cotyledons and
senescent leaves (Fig 3f & i) raises the interesting possibil-
ity that CAT2 may also play a role in glyoxysomal removal,
as well as CAT1. On the other hand, while CAT3, a Class II
catalase, was predominantly expressed in vascular tissues, it
was also expressed in mesophyll cells adjacent to the vas-
culature (Fig 3m & o). Thus, it is possible that CAT3 may be
active in these tissues as well as CAT2. These results suggest
that although CAT2 is more important in the removal of
H2O2 produced during photorespiration, there is some func-
tional redundancy with CAT1 and CAT3. Our approach to
studying the components of catalase isoforms provides a
valuable tool for further research into the functions of the
catalase family.
ACKNOWLEDGMENTS
This work was supported in part by Major State Basic
Research Program (2007CB108701), National Natural
Science Foundation of China.
REFERENCES
Apel K. & Hirt H. (2004) Reactive oxygen species: metabolism,
oxidative stress, and signal transduction. Annual Review of Plant
Biology 55, 373–399.
Atherton E. & Sheppard R.C. (1989) Solid Phase Peptide Synthesis:
A Practical Approach. IRL Press, Oxford.
Baker C.J. & Orlandi E.W. (1995) Active oxygen in plant patho-
genesis. Annual Review of Phytopathology 33, 299–321.
Beevers H. (1982) Glyoxysomes in higher plants. Annals of the
New York Academy of Sciences 386, 243–253.
Bradford M.M. (1976) A rapid and sensitive method for the quan-
titation of microgram quantities of protein utilizing the principle
of protein-dye binding. Analytical Biochemistry 72, 248–254.
Bueso E., Alejandro S., Carbonell P., Perez-Amador M.A., Fayos J.,
Belles J.M., Rodriguez P.L. & Serrano R. (2007) The lithium
tolerance of the Arabidopsis cat2 mutant reveals a cross-talk
between oxidative stress and ethylene. The Plant Journal 52,
1052–1065.
Clare D.A., Duong M.N., Darr D., Archibald F. & Fridovich I.
(1984) Effects of molecular oxygen on detection of superoxide
radical with nitroblue tetrazolium and on activity stains for cata-
lase. Analytical Biochemistry 140, 532–537.
Clough S.J. & Bent A.F. (1984) Floral dip: a simplified method for
Agrobacterium -mediated transformation of Arabidopsis
thaliana. The Plant Journal 16, 735–743.
Corpas F.J., Barroso J.B. & del Río L.A. (2001) Peroxisomes as a
source of reactive oxygen species and nitric oxide signal mol-
ecules in plant cells. Trends in Plant Science 6, 145–150.
Dat J., Vandenabeele S., Vranová E, Van Montagu M., Inzé D. &
Van Breusegem F. (2000) Dual action of the active oxygen
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
species during plant stress responses. Cellular and Molecular
Life Sciences 57, 779–795.
Du Y.Y., Wang P.C., Chen J. & Song C.P. (2008) Comprehensive
functional analysis of the catalase gene family in Arabidopsis
thaliana. Journal of Integrative Plant Biology 50, 1318–1326.
Feierabend J. (2005) Catalases in plants: molecular and functional
properties role in stress defence. In Antioxidants and Reactive
Oxygen Species in Plants (ed. N. Smirnoff), pp. 101–140. Black-
well Publishing, Oxford.
Fita I. & Rossmann M.G. (1985) The active center of catalase.
Journal of Molecular Biology 185, 21–37.
Foyer C.H. & Noctor G. (2005) Redox homeostasis and antioxidant
signaling: a metabolic interface between stress perception and
physiological responses. The Plant Cell 17, 1866–1875.
Foyer C.H., Bloom A.J., Queval G. & Noctor G. (2009) Photores-
piratory metabolism: genes, mutants, energetics, and redox
signaling. Annual Review of Plant Biology 60, 455–484.
Friedrich J.W. & Huffaker R.C. (1980) Photosynthesis, leaf resis-
tances, and ribulose-1,5-bisphosphate carboxylase degradation
in senescing barley leaves. Plant Physiology 65, 1103–1107.
Frugoli J.A., Zhong H.H., Nuccio M.L., McCourt P., McPeek M.A.,
Thomas T.L. & McClung C.R. (1996) Catalase is encoded by a
multigene family in Arabidopsis thaliana (L.) Heynh. Plant
Physiology 112, 327–336.
Fukamatsu Y., Yabe N. & Hasunuma K. (2003) Arabidopsis NDK1
is a component of ROS signaling by interacting with three cata-
lases. Plant & Cell Physiology 44, 982–989.
Gechev T.S., Van Breusegem F., Stone J.M., Denev I. & Laloi C.
(2006) Reactive oxygen species as signals that modulate plant
stress responses and programmed cell death. BioEssays: News
and Reviews in Molecular, Cellular and Developmental Biology
28, 1091–1101.
Gillham D.J. & Dodge A.D. (1986) Hydrogen peroxide-scavenging
systems with pea chloroplasts. A quantitative study. Planta 167,
246–251.
Gouet P., Jouve H.M. & Dideberg O. (1995) Crystal structure of
Proteus mirabilis PR catalase with and without bound NADPH.
Journal of Molecular Biology 249, 933–954.
Grzybowska E.A., Wilczynska A. & Siedlecki J.A. (2001) Regula-
tory functions of 3′UTRs. Biochemical and Biophysical Research
Communications 288, 291–295.
Heinze M. & Gerhardt B. (2002) Plant catalase. In Plant Peroxi-
somes: Biochemistry, Cell Biology and Biology and Biotechno-
logical Application (eds A. Baker & I.A. Graham), pp. 103–140.
Kluwer Academic Publisher, Dordrecht.
Jefferson R.A. (1987) Assaying chimeric genes in plants: the GUS
Gene Fusion System. Plant Molecular Biology Reporter 5, 387–
405.
Jiang C.Z., Rodermel S.R. & Shibles R.M. (1993) Photosynthesis,
rubisco activity and amount, and their regulation by transcrip-
tion in senescing soybean leaves. Plant Physiology 101, 105–
112.
Joo J.H., Bae Y.S. & Lee J.S. (2001) Role of auxin-induced reactive
oxygen species in root gravitropism. Plant Physiology 126, 1055–
1060.
Kamada T., Nito K., Hayashi H., Mano S., Hayashi M. & Nishimura
M. (2003) Functional differentiation of peroxisomes revealed by
expression profiles of peroxisomal genes in Arabidopsis thaliana.
Plant & Cell Physiology 44, 1275–1289.
Karpinski S., Gabrys H., Mateo A., Karpinska B. & Mullineaux
P.M. (2003) Light perception in plant disease defence signalling.
Current Opinion in Plant Biology 6, 390–396.
Kendall A.C., Keys A.J., Turner J.C., Lea P.J. & Miflin B.J. (1983)
The isolation and characterisation of a catalase-deficient mutant
of barley (Hordeum vulgare L.). Planta 159, 505–511.
Kwak J.M., Mori I.C., Pei Z.M., Leonhardt N., Torres M.A., Dangl

J.L., Bloom R.E., Bodde S., Jones J.D. & Schroeder J.I. (2003)
NADPH oxidase AtrbohD and AtrbohF genes function in ROS-
dependent ABA signaling in Arabidopsis. The EMBO Journal
22, 2623–2633.
Li R.J., Hua W. & Lu Y.T. (2006) Arabidopsis cytosolic glutamine
synthetase AtGLN1;1 is a potential substrate of AtCRK3
involved in leaf senescence. Biochemical and Biophysical
Research Communications 342, 119–126.
Loprasert S., Vattanaviboon P., Praituan W., Chamnongpol S. &
Mongkolsuk S. (1996) Regulation of the oxidative stress protec-
tive enzymes, catalase and superoxide dismutase in Xanthomo-
nas – a review. Gene 179, 33–37.
McClung C.R. (1997) Regulation of catalases in Arabidopsis. Free
Radical Biology & Medicine 23, 489–496.
Michelet L., Zaffagnini M., Marchand C. et al. (2005) Glutathiony-
lation of chloroplast thioredoxin f is a redox signaling mecha-
nism in plants. Proceedings of the National Academy of Sciences
102, 16478–16483.
Michelet L., Zaffagnini M., Vanacker H., Le Marechal P., Marchand
C., Schroda M., Lemaire S.D. & Decottignies P. (2008) In vivo
targets of S-thiolation in Chlamydomonas reinhardtii. Journal of
Biological Chemistry 283, 21571–21578.
Murata Y., Pei Z.M., Mori I.C. & Schroeder J. (2001) Abscisic acid
activation of plasma membrane Ca(2+) channels in guard cells
requires cytosolic NAD(P)H and is differentially disrupted
upstream and downstream of reactive oxygen species production
in abi1-1 and abi2-1 protein phosphatase 2C mutants. The Plant
Cell 13, 2513–2523.
Ni W. & Trelease R.N. (1991) Post-Transcriptional Regulation of
Catalase Isozyme Expression in Cotton Seeds. The Plant Cell 3,
737–744.
Nishimura M., Hayashi M., Kato A., Yamaguchi K. & Mano S.
(1996) Functional transformation of microbodies in higher plant
cells. Cell Structure and Function 21, 387–393.
Noctor G., Veljovic-Jovanovic S., Driscoll S., Novitskaya L. & Foyer
C.H. (2002) Drought and oxidative load in the leaves of C3
plants: a predominant role for photorespiration? Annals of
Botany 89, 841–850.
Orendi G., Zimmermann P., Baar C. & Zentgraf U. (2001) Loss of
stress-induced expression of catalase3 during leaf senescence in
Arabidopsis thaliana is restricted to oxidative stress. Plant
Science 161, 301–314.
Proudfoot N.J., Furger A. & Dye M.J. (2002) Integrating mRNA
processing with transcription. Cell 108, 501–512.
Queval G., Issakidis-Bourguet E., Hoeberichts F.A., Vandorpe M.,
Gakiere B., Vanacker H., Miginiac-Maslow M., Van Breusegem
F. & Noctor G. (2007) Conditional oxidative stress responses in
the Arabidopsis photorespiratory mutant cat2 demonstrate that
redox state is a key modulator of daylength-dependent gene
expression, and define photoperiod as a crucial factor in the
regulation of H2O2-induced cell death. The Plant Journal 52,
640–657.
Queval G., Thominet D., Vanacker H., Miginiac-Maslow M.,
Gakiere B. & Noctor G. (2009) H2O2-activated up-regulation of
glutathione in Arabidopsis involves induction of genes encoding
enzymes involved in cysteine synthesis in the chloroplast.
Molecular Plant 2, 344–356.
Sambrook J., Fritsch E.F. & Maniatis T. (1989) Molecular Cloning:
A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY.
Santos I., Pires H., Almeida J.M., Fidalgo F., Confraria A., Duarte
M., Borlido J. & Salema R. (2006) Phylogenetic relationship of
potato CAT1 and CAT2 genes, their differential expression in
non-photosynthetic organs and during leaf development, and
their association with different cellular processes. Functional
Plant Biology 33, 639–651.
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
Characterization of Arabidopsis catalase genes 1669
Scandalios J.G., Tong W.F. & Roupakias D.G. (1980) Cat3, a third
gene locus coding for a tissue-specific catalase in maize: genetics,
intracellular location, and some biochemical properties. Molecu-
lar and General Genetics 179, 33–41.
Scandalios J.G. (1990) Regulation and properties of plant catalase.
In Causes of Photooxidative Stress and Amelioration of Defense
Systems in Plants (eds C.H. Foyer & P.M. Mullineaux), pp. 275–
315. CRC Press, Boca Raton, FL.
Schmidt M., Dehne S. & Feierabend J. (2002) Post-transcriptional
mechanisms control catalase synthesis during its light-induced
turnover in rye leaves through the availability of the hemin
cofactor and reversible changes of the translation efficiency of
mRNA. Plant Journal 31, 601–613.
Schmidt M., Grief J. & Feierabend J. (2006) Mode of translational
activation of the catalase (cat1) mRNA of rye leaves (Secale
cereale L.) and its control through blue light and reactive oxygen.
Planta 223, 835–846.
Smith I.K., Kendall A.C., Keys A.J., Turner J.C. & Lea P.J. (1985)
The regulation of the biosynthesis of glutathione in leaves of
barley (Hordeum vulgare L.). Plant Science 41, 11–17.
Suzuki M., Miyamoto R., Hattori T., Nakamura K. & Asahi T.
(1995) Differential regulation of the expression in transgenic
tobacco of the gene for beta-glucuronidase under the control of
the 5′-upstream regions of two catalase genes from castor bean.
Plant & Cell Physiology 36, 273–279.
Tatusova T.A. & Madden T.L. (1999) Blast 2 sequences, a new tool
for comparing protein and nucleotide sequences. FEMS Micro-
biology Letters 174, 247–250.
Vanacker H., Carver T.L. & Foyer C.H. (2000) Early H2O2 accu-
mulation in mesophyll cells leads to induction of glutathione
during the hyper-sensitive response in the barley-powdery
mildew interaction. Plant Physiology 123, 1289–1300.
Vandenabeele S., Vanderauwera S., Vuylsteke M., Rombauts S.,
Langebartels C., Seidlitz H.K., Zabeau M., Van Montagu M.,
Inzé D. & Van Breusegem F. (2004) Catalase deficiency drasti-
cally affects gene expression induced by high light in Arabidopsis
thaliana. The Plant Journal 39, 45–58.
Vanderauwera S., Zimmermann P., Rombauts S., Vandenabeele S.,
Langebartels C., Gruissem W., Inzé D. & Van Breusegem F.
(2005) Genome-wide analysis of hydrogen peroxide-regulated
gene expression in Arabiodopsis reveals a high light-induced
transcriptional cluster involved in anthocyanin biosynthesis.
Plant Physiology 139, 806–821.
Veljovic-Jovanovic S.D., Pignocchi C., Noctor G. & Foyer C.H.
(2001) Low ascorbic acid in the vtc-1 mutant of Arabidopsis is
associated with decreased growth and intracellular redistri-
bution of the antioxidant system. Plant Physiology 127, 426–
435.
Verslues P.E., Batelli G., Grillo S., Agius F., Kim Y.S., Zhu J.,
Agarwal M., Katiyar-Agarwal S. & Zhu J.K. (2007) Interaction
of SOS2 with nucleoside diphosphate kinase 2 and catalases
reveals a point of connection between salt stress and H2O2 sig-
naling in Arabidopsis thaliana. Molecular and Cellular Biology
27, 7771–7780.
Willekens H., Langebartels C., Tire C., Van Montagu M., Inzé D. &
Van Camp W. (1994) Differential expression of catalase genes in
Nicotiana plumbaginifolia (L.). Proceedings of the National
Academy of Sciences of the United States of America 91, 10450–
10454.
Willekens H., Inzé D. & Van Montagu M. (1995) Catalases in
plants. Molecular Breeding 1, 207–208.
Willekens H., Chamnongpol S., Davey M., Schraudner M.,
Langebartels C., Van Montagu M., Inze D. & Van Camp W.
(1997) Catalase is a sink for H2O2 and is indispensable for
stress defence in C3 plants. The EMBO Journal 16, 4806–
4816.
1670 Y-Q. Hu et al.

Xing Y., Jia W. & Zhang J. (2007) AtMEK1 mediates stress- Received 10 December 2009; received in revised form 23 April 2010;
induced gene expression of CAT1 catalase by triggering H2O2 accepted for publication 25 April 2010
production in Arabidopsis. Journal of Experimental Botany 58,
2969–2981.
Xing Y., Jia W. & Zhang J. (2008) AtMKK1 mediates ABA-
induced CAT1 expression and H2O2 production via
SUPPORTING INFORMATION
AtMPK6-coupled signaling in Arabidopsis. The Plant Journal 54, Additional Supporting Information may be found in the
440–451.
online version of this article:
Zhao J., Hyman L. & Moore C. (1999) Formation of mRNA 3′ ends
in eukaryotes: mechanism, regulation, and interrelationships Figure S1. Illustration of method of constructing chimeric
with other steps in mRNA synthesis. Microbiology and Molecu- gene CAT1-2 and CAT2-1.
lar Biology Reviews 63, 405–445.
Table S1. Synthetic oligonucleotide primers used in this
Zhong H.H., Resnick A.S., Straume M. & Robertson McClung C.
(1997) Effects of synergistic signaling by phytochrome A and study.
cryptochrome1 on circadian clock-regulated catalase expression. Please note: Wiley-Blackwell are not responsible for the
The Plant Cell 9, 947–955.
content or functionality of any supporting materials sup-
Zimmermann P., Heinlein C., Orendi G. & Zentgraf U. (2006)
Senescence-specific regulation of catalases in Arabidopsis plied by the authors. Any queries (other than missing mate-
thaliana (L.) Heynh. Plant, Cell & Environment 29, 1049– rial) should be directed to the corresponding author for the
1060. article.

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