Journal of Integrative Plant Biology 2008, 50 (10): 1318–1326

Comprehensive Functional Analysis of the Catalase Gene

Family in Arabidopsis thaliana
∗
Yan-Yan Du1,2 , Peng-Cheng Wang2 , Jia Chen1 and Chun-Peng Song2
(1 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China;
2
Laboratory of Plant Stress Biology, Department of Biology, Henan University, Kaifeng 475001, China)

Abstract

In Arabidopsis, catalase (CAT) genes encode a small family of proteins including CAT1, CAT2 and CAT3, which catalyze
the decomposition of hydrogen peroxide (H 2 O 2 ) and play an important role in controlling homeostasis of reactive oxygen
species (ROS). Here, we analyze the expression profiles and activities of three catalases under different treatments including
drought, cold, oxidative stresses, abscisic acid and salicylic acid in Arabidopsis. Our results reveal that CAT1 is an important
player in the removal of H 2 O 2 generated under various environmental stresses. CAT2 and CAT3 are major H 2 O 2 scavengers
that contribute to ROS homeostasis in light or darkness, respectively. In addition, CAT2 is activated by cold and drought
stresses and CAT3 is mainly enhanced by abscisic acid and oxidative treatments as well as at the senescence stage. These
results, together with previous data, suggest that the network of transcriptional control explains how CATs and other
scavenger enzymes such as peroxidase and superoxide dismutase may be coordinately regulated during development,
but differentially expressed in response to different stresses for controlling ROS homeostasis.

Key words: catalase; isozymes; reactive oxygen species; stress; transcription.

Du YY, Wang PC, Chen J, Song CP (2008). Comprehensive functional analysis of the catalase gene family in Arabidopsis thaliana. J. Integr. Plant
Biol. 50(10), 1318–1326.

Available online at www.jipb.net

Catalase is a common enzyme found in nearly all living
organisms, and consists of tetrameric iron porphyrin protein. Its
function is to catalyze the decomposition of hydrogen peroxide
(H 2 O 2 ) to water and oxygen. In animal cells, catalase is encoded
by a unique gene; however, a small gene family encodes
catalase in plants. For example, the catalase gene family has
been found to encode three members including CAT1, CAT2
and CAT3 in Arabidopsis genomes (Frugoli et al. 1996). Many
documents have demonstrated the function, cellular localization
and expression profiles of catalase in some plant organisms
(Guan and Scandalios 2000a, 2000b, 2002).
Several studies in Arabidopsis reveal that expression of CAT2
and CAT3 is controlled by circadian rhythm, with a morning-
Received 18 Feb. 2008 Accepted 17 Jun. 2008
Supported by the National Natural Science Foundation of China (30530430
and 30625005).
∗
Author for correspondence.
Tel: +86 37 8388 0007;
Fax: +86 37 8388 0006;
E-mail: <songcp@henu.edu.cn>.

C 2008 Institute of Botany, the Chinese Academy of Sciences
doi: 10.1111/j.1744-7909.2008.00741.x
specific phase for CAT2 and an evening-specific phase for
CAT3 (Zhong and McClung 1996; Michael and McClung 2002).
CAT2 expression is downregulated during leaf senescence,
whereas CAT3 expression is induced by age and senescence
(Zimmermann et al. 2006; Xing et al. 2007). Interestingly, the
mRNA abundance of CAT is also accumulated under drought,
abscisic acid (ABA) and salt treatments (Xing et al. 2007). Also,
stress-induced expression of CAT1 depends on the production
of H 2 O 2 , which is triggered by a mitogen-actived protein kinase
(MAPK) kinase AtMEK1 (Xing et al. 2007). These data suggest
that various environmental stresses always enhance the tran-
scription of CATs, and subsequently increase enzyme activity of
catalase, leading to redox homeostasis in plant cells. However,
tissue- and/or stress-specific expression profiles of CAT genes
are still largely unknown.
In this research, we analyze the expression profiles and
activities of three catalases under different treatments including
drought, cold, oxidative stresses, ABA and salicylic acid (SA)
in Arabidopsis. Our results suggest that expression of three
CAT genes is indeed enhanced with tissue- and stress-specific
manners. Moreover, the activity level of each CAT is also similar
to those observed in gene expression analysis. This information
may be valuable for further analysis of the profile and function
diversity of catalase.
 Comprehensive Functional Analysis of Catalases 1319

Results rosette leaves and siliques. However, expression of CAT1 is low

Expression patterns of catalase genes in different tissues
To determine the expression patterns of catalase genes in
Arabidopsis, we fused the promoter region of catalase genes
to the β-glucuronidase (GUS) reporter gene and detected GUS
activity in transgenic plants. As shown in Figure 1A, the GUS
activity in CAT1::GUS plants is mainly found in cotyledon,
in bolts, roots and true leaves. Unlike CAT1, the CAT2 promoter
is highly activated in germinating seeds, cotyledon and young
leaves, bolts, siliques and flowers. Interestingly, the expression
of CAT2 is dramatically decreased in 6-week-old plants as
compared with the seedlings (Figure 1B). CAT3 is ubiquitously
expressed, having a stronger expression in roots at different
developmental stages and young leaves (Figure 1C). These
results suggest that CAT1 and CAT2 are mainly expressed

Figure 1. The tissue-specific expression of catalase genes in Arabidopsis.

(A, B, C) The β-glucuronidase staining of transgenic plants containing pCAT1::GUS, pCAT2::GUS and pCAT3::GUS construct, respectively. I to VII
show different developmental stages and different tissues. I, 2-day-old seedlings; II, 7-day-old seedlings; III, 2-week-old seedlings; IV, 6-week-old
plants; V, stems; VI, inflorescences; VII, siliques.
(D) The expression levels of CAT1 (), CAT2 () and CAT3 (. . .) in different tissues in Arabidopsis. The mean value of three replicates was normalized
using UBQ10 as the internal control.
1320 Journal of Integrative Plant Biology Vol. 50 No. 10
in leaves and siliques, whereas CAT3 is mainly expressed in
stems and roots.
To confirm the tissue-expression patterns of CAT genes,
we also carried out real-time polymerase chain reaction (PCR)
analysis to detect the mRNA abundance of catalase genes in
different tissues. After normalization to the internal control gene
UBQ10, mRNA levels of CATs are almost similar to the GUS ac-
tivity analysis. For example, CAT3 expression intensity in stems
is higher than that of CAT2. Similarly, the strong expression of
CAT1 and CAT2 is observed in leaves and siliques; however,
very high levels of CAT2 are detected in flowers. In addition, the
expression patterns of CAT genes in rosette leaves are different
from results obtained by GUS staining. This is probably because
the expression of catalase genes is sensitive to growth stage,
light condition and senescence (Zimmermann et al. 2006), and
slight variations in developmental stages and growth conditions
may result in different expression profiles.
Transcription and enzyme activity of catalases are
regulated by abiotic stresses
To determine whether the expression of endogenous CAT
genes is affected by environmental conditions, mRNA from
Arabidopsis plants treated with cold, drought, oxidative stresses,
including 3-aminotriazol (3-AT), methyl viologen (MV), H 2 O 2 ,
and the plant hormones SA and ABA were analyzed by quanti-
tative real-time PCR. There are high basal levels of transcripts
in CAT2 and CAT3 under non-stressed conditions (Figure 2A).
Transcription of CAT2 is upregulated in the first 3 h, and then
declines dramatically. On the contrary, the expression of CAT3
is obviously downregulated in the first 2 h, and after that a
gradual increase is observed. In addition, the expression of
CAT1 is very weak and no significant variation is detected.
Under stress conditions, however, expression of CAT genes
is induced (Figure 2B–H). In all treatments, the mRNA abun-
dance of CAT1 is significantly increased. For example, the ex-
pression of CAT1 is rapidly induced and reaches a peak after 1-h
treatment under dehydration (Figure 2D); whereas the highest
transcription activity always appears at 2 or 3 h after treatments
with cold, oxidative stresses and ABA. However, the transcript
levels of CAT1 are only slightly enhanced by SA treatment
(Figure 2C). Treatment with dehydration and 20 mmol/L H 2 O 2
for 1 and 6 h, respectively, significantly increases transcription
accumulation of CAT2. Interestingly, transcription of CAT3 is
distinctly enhanced with different expression patterns under
ABA and oxidative treatments. Alterations in the transcript levels
from the CAT3 gene are too low to be seen clearly under our
other stress treatments such as cold, SA and drought.
To further examine whether abiotic stresses and hormones
affect the catalase activity, we extracted total proteins from
seedlings treated with ABA, SA, cold, drought, 3-AT, MV and
H 2 O 2 for 3 h. As shown in Figure 2I, almost all of the treatments
2008
can enhance catalase activity, except SA and 3-AT. In tobacco,
SA can bind to CAT2, and repress catalase activity (Chen et al.
1993). 3-AT is a potent inhibitor of CAT (Gechev et al. 2002);
application of 3-AT to Arabidopsis plants dramatically reduces
total CAT activity.
Isolation and characterization of CAT-deficient
mutant plants
The T-DNA and RNAi lines are then used to explore the contribu-
tions of the CAT1-3 genes to catalase activity to catalyze H 2 O 2
decomposition. For CAT2, a homozygous T-DNA insertion line
(salk_076998) was detected by PCR. For another two catalase
genes, two independent RNAi lines (cat1-01 and cat1-04; and
cat3-03 and cat3-09) were randomly chosen and confirmed
by real-time PCR. As shown in Figure 3A, the expression of
CAT1 in cat1-01 and cat1-04, and CAT3 in cat3-03 and cat3-
09 are knocked down, respectively, and the transcript levels of
two other catalase genes are not affected in CAT1 or CAT3
RNAi lines. Interestingly, the expression of CAT1 and CAT3
dramatically increased in cat2-1 T-DNA mutants, indicating that
there is a feedback loop to keep H 2 O 2 homeostasis in cells
through transcriptional regulation of CAT genes. To examine
the activity of each catalase under stresses, the isozymes of
catalase were determined in native PolyAcrylamide Gel Elec-
trophoresis (PAGEs) after specific activity staining. As shown
in Figure 3B, the activities of CAT1, CAT2 and CAT3 in these
mutants were indeed decreased. In the cat1 RNAi lines, overall
catalase activities were decreased by 8%. Only 27% and 45% of
CAT activity, however, were measured in cat2 and cat3 mutants,
respectively (Figure 3C). Consistent with the expression results
in Figure 2A, we conclude that CAT2 and CAT3 are the major
catalases under normal conditions.
Zymogram analysis of catalase activity
under abiotic stresses
To further investigate the regulation of catalase activity in
response to hormone and abiotic stresses, we carried out the
zymogram analysis of catalase activities using wild type and
CAT-deficient Arabidopsis seedlings treated with ABA, cold,
drought, H 2 O 2 and MV. As shown in Figure 4E, after 3-h
cold treatment, the activity of CAT2 is significantly increased;
whereas CAT1 and CAT3 are only slightly enhanced. These
results suggest that CAT2 plays a major role in the cold stress
response. Consistent with this, when CAT2 is absent, activities
of CAT1 and CAT3 are improved as a complement. In CAT1
or CAT3 RNAi lines, the absence of CAT1 and CAT3 does not
affect cold-induced catalase activities. On the contrary, both
ABA and MV treatment only activate the activities of CAT1 and
CAT3. When seedlings are treated with H 2 O 2 , the activities
of all three catalases are obviously enhanced. These data
 Comprehensive Functional Analysis of Catalases 1321

Figure 2. Expression patterns of three CAT genes and changes in catalase (CAT) activities under different environmental stresses.

(A) Transcript levels of CAT1 (), CAT2 (◦) and CAT3 () in control conditions.
(B–H) Transcript levels of CAT1 (), CAT2 (◦) and CAT3 () under different stress conditions. The mean value of three replicates was normalized
using UBQ10 as the internal control. See Material and Methods for details.
(I) The total activities of CAT from seedlings treated with stress conditions or hormones.

suggest that there exists a feedback loop in controlling the
reactive oxygen species (ROS) homeostasis. Drought stress
also enhances the activity of all three catalases, with the CAT2
increased most obviously.
H 2 O 2 concentration increases obviously in WT and three CAT-
deficient plants, whereas the concentration of H 2 O 2 in cat2-1 is
much higher than that of other plants (Figure 5B). These data
clearly indicate that the deficiency of CAT2 decreases the H 2 O 2
scavenging ability under cold treatment.

Detection of H 2 O 2 production in CAT-deficient plants

The H 2 O 2 concentration in wild type and CAT-deficient plants Discussion


were detected with 3,3 -diaminobenzidine tetrachloride (DAB)
staining. As shown in Figure 5A, the accumulation of H 2 O 2 is
not induced and the low basal levels in the deficient mutants of
CATs do not differ much from that of the wild-type plants under
control conditions. However, after cold treatment for 2 h, the
Reactive oxygen species have been reported as toxic and
signal molecules in plant responsive to environmental stresses
(Neill et al. 2002; Laloi et al. 2004; Cheng and Song 2006;
Gechev 2006). Thus, there must be a fine mechanism for its
1322 Journal of Integrative Plant Biology Vol. 50 No. 10 2008

Figure 3. Expression and protein activities of catalase (CAT) in CAT-deficient plants.

(A) Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) results showing CAT1 (), CAT2 () and CAT3 (. . .) expression
in wild type, cat2 T-DNA insertion line, cat1 or cat3 RNAi lines, respectively. UBQ10 primers were used in the PCR reaction as an internal control.
(B) Catalase zymogram showing CAT activities in wild type and CAT-deficient mutants.
(C) Quantitative analysis for total CAT activities in wild type (WT) and CAT-deficient mutants, showing the decreases of CAT activity in cat1, cat2 and
cat3 mutants.

metabolism and synthesis to control the homeostasis of ROS in
plant cells. Here we investigated the transcription of CAT genes
and the activity of catalase in Arabidopsis seedlings treated with
different abiotic stresses or hormones. These results reveal
that different stresses may trigger distinct signal transduction
pathways and activate transcription of different CAT genes.
Lots of evidence suggests that the expression and activities
of catalase are always activated by stresses in Arabidopsis
(Williamson and Scandalios 1992; Guan and Scandalios 2000b;
Orendi et al. 2001; Vandenabeele et al. 2004; Luna et al.
2005; Verslues et al. 2007; Xing et al. 2007). CAT1 is found
to exhibit very low levels of transcription and enzyme activity in
normal conditions (Figures 2A, 3B). However, all of the stresses
that we have examined can induce the expression of CAT1
and dramatically increase the enzyme activities of catalase.
These data indicate that the CAT1 or CAT2 may act as main
scavengers for H 2 O 2 in response to the stresses and play an
important role in adapting to environmental stresses. However,
abiotic stresses activate the expression and activity of CAT1,
which may act as a feedback regulating of ROS signaling.
CAT2 has been known as a light-induced catalase gene and
its expression shows a circadian rhythm with a maximum in the
early morning (Zhong et al. 1994; Zhong and McClung 1996). A
previous report has indicated that deficiency of CAT2 reduced
resistance to high light in Arabidopsis plants (Vanderauwera
et al. 2005). Consistent with this, CAT2 mainly expresses in
photosynthesis tissues including leaves (Figure 1B). In addition,
the expression levels of CAT2 are higher than those of CAT1
and CAT3 under non-stressed conditions (Figure 2A), and
activities of CAT2 are improved distinctly in drought, cold and
oxidative stresses (Figure 4). Furthermore, the T-DNA insertion
in CAT2 results in a substantial reduction in catalase activity
(Figure 3C) and more H 2 O 2 accumulation of leaves under cold
treatment (Figure 5B). On the basis of the results presented here
as well as those reported previously (Zhong and McClung 1996;
Vanderauwera et al. 2005), we suggest that CAT2 can consti-
tutively contribute catalase activity in normal conditions, and
also plays important roles in drought and cold stress responses
(McClung et al. 2000; Queval et al. 2007). On the contrary,
expression of CAT3 is always activated under darkness, and
accumulated in senescence leaves (Figure 1C). In cat2-1, the
expression and activity of CAT3 increase obviously under stress
treatments. These data suggest that CAT3 may be involved
in the regulation of oxidative homeostasis during senescence
(Park et al. 1998; Zimmermann et al. 2006).
In summary, our data about functional analysis of catalase
gene family under abiotic stresses reveal the general functional
diversity of catalases in Arabidopsis. CAT1 is an important
player in the removal of H 2 O 2 generated under various envi-
ronmental stresses. CAT2 and CAT3 are major H 2 O 2 scav-
engers to contribute to ROS homeostasis in light or darkness,
respectively. In addition, CAT2 is activated by cold and drought
stresses, and CAT3 is mainly enhanced by ABA and oxidative
treatments as well as at the senescence stage. This network
of transcriptional control also explains how CATs and other
scavenger enzymes such as peroxidase and superoxide dis-
mutase may be coordinately regulated during development, but
differentially expressed in response to different stresses.
Materials and Methods
Plant materials and treatments
Wild type, mutant and transgenic Arabidopsis seedlings were
grown on Murashige-Skoog (MS) medium (3% sucrose and
 Comprehensive Functional Analysis of Catalases 1323

Figure 4. Changes in catalase (CAT) activities under abiotic stresses and abscisic acid (ABA) treatment.

(A, C, E, G, I) CAT activity staining in wild type (WT) and CAT-deficient lines treated with ABA, drought, cold, H 2 O 2 and methyl viologen (MV).
(B, D, F, H, J) Total CAT activities in WT and CAT-deficient lines treated with ABA (), drought (), cold (), H 2 O 2 () and MV (). The proteins
from plants without treatments were used as controls ().
1324 Journal of Integrative Plant Biology Vol. 50 No. 10
Figure 5. Changes in H 2 O 2 concentration in wild type (WT) and CAT-
deficient lines treated with cold stress.

WT and CAT-deficient mutant seedlings were stained with 3,3 -
diaminobenzidine tetrachloride (DAB) to show the changes of H 2 O 2
concentration under control (A) and cold treatment (B).
0.6% agar) for 14 d under a light:dark cycle of 16:8 h. For
the hormone and oxidative stress treatments, seedlings were
sprayed with 100 μmol/L ABA, 10 μmol/L SA, 20 mmol/L
hydrogen peroxide, 10 μmol/L methyl viologen, 20 mmol/L 3-
aminotriazole, respectively. For the cold treatment, seedlings
on agar plates were transferred to 4o C under light. The drought-
treated seedlings were desiccated in the growth chamber. The
whole seedlings under treatment for 0, 1, 2, 3 and 6 h were
frozen in liquid nitrogen.
T-DNA insertion line of CAT2 gene (Salk_076998) was
obtained from the Arabidopsis Biological Resource Center
(ABRC). The homozygous mutant plants were confirmed by

PCR using gene-specific primers (LP, 5 -ACATTTTGGAGCAT
  
TGACTGG-3 ; RP, 5 -TCTGGTGCTCCTGTATGGAAC-3 ) and

left border primers (LBal, 5 -TGGTTCACGTAGTGGGCCATC
  
G-3 ; LBb1, 5 -GCGTGGACCGCTTGCTGCAACT-3 ).
RNAi transgenic plants of CAT1 and CAT3 were generated
by following the procedures as described by Song et al. (2005).
The cDNA fragments of CAT1 and CAT3 were amplified by PCR
with the following primer pairs: for CAT1, forward primer 5 -
GCCGGATCCAGGCGCGCCTATGACTCGATCGCAGCCG-3
and reverse primer 5 -GCACCATGGTCTCTAGAAGACGGTG
TCTCTGACTA-3 , for CAT3, forward primer 5 -CAGGATCCA
GGCGCGCCATTCTACACCACA-3 and reverse primer 5 -
AATCTAGACCCATGGAAACGGACAATAACC-3 . The result-
ing PCR products were digested with Asc I and Nco I and ligated
to the Asc I-Nco I-cleaved pFGC5941 vector. The generated
constructs contained sense gene fragments. PCR products
were also digested with BamH I and Xba I and inserted into the
BamH I-Xba I sites of the template plasmid, to complete the
inverted repeat construct. The double-strand RNA constructs
2008
were introduced into the Agrobacterium tumefaciens strain
LBA4404 and transformed into wild-type A. thaliana (ecotype
Columbia) plants by floral infiltration.
GUS activity assays
Regions of approximately 1500-bp upstream of the CAT1, CAT2
and CAT3 translational starts were amplified from the Col-0 ge-
nomic DNA by PCR with oligonucleotides incorporating Sal I and
BamH I sites. PCR fragments were cloned into the Sal I-BamH I
site of the promoterless β-glucuronidase (GUS) expression vec-
tor pCAMBIA1381. The constructs pCAT1::GUS, pCAT2::GUS
and pCAT3::GUS were transferred from Escherichia coli into
A. tumefaciens LBA4404, respectively. Arabidopsis plants were
transformed by floral infiltration with A. tumefaciens containing
the three constructs. Histochemical localizations of GUS ac-
tivities in the transgenic plants were analyzed after incubating
the transgenic plants in X-gluc buffer (50 mmol/L sodium phos-
phate buffer, pH 7.0, 10 mmol/L ethylenediaminetetraacetic acid
[EDTA], 0.1% Triton X-100, 0.5 mmol/L potassium ferrocyanide,
and 2 mg/mL 5-bromo-4-chloro-3-indolyl glucuronide) at 37o C
for 6 h.
Detection of catalase activities and isozymes analysis
Total protein of Arabidopsis seedlings was extracted and de-
termined as described previously (Clarke et al. 2002). Briefly,
0.2 g of seedlings under various treatments were ground to
a fine powder in liquid nitrogen and homogenized in 1 mL of
extraction buffer (100 mmol/L Tris-HCl, pH 8.0, 20% glycerol
and 30 mmol/L dithiothreitol). After centrifugation at 12 000g
for 15 min at 4o C, the protein contents of supernatant were
quantified according to the method of Bradford (1976) using
bovine serum albumin (BSA) as a standard. For catalase
activities assays, 150 μg of total proteins were added to 3.0 mL
buffered substrate (50 mmol/L potassium phosphate, pH 7.0
and 20 mmol/L H 2 O 2 ). The decomposition of H 2 O 2 was fol-
lowed by the decline in absorbance at 240 nm. The activity is
expressed in units, where one unit of catalase converts 1 μmol
of H 2 O 2 /min.
For the analysis of CAT isozymes, the protein extracts con-
taining 20 μg of total protein were loaded and separated in
native polyacrylamide gels according to Zimmermann et al.
(2006). After electrophoresis, the catalase activity was assayed
according to Chandlee and Scandalios (1983). Briefly, the gels
were soaked in 0.01% of H 2 O 2 solution for 5 min, washed twice
with double-distilled water and incubated for 30min in 1% FeCl 3
and 1% K 3 Fe(CN) 6 . After staining, the gels were then washed
twice in water and photographed.
Detection of H 2 O 2 concentration in plant
The detection of H 2 O 2 in tissues was carried out according
to Thordal-Christensen et al. (1997). Two-week-old seedlings

were treated with cold, drought, or sprayed with H 2 O 2 , or chilling
in 4 o C for 2 h, then were incubated for 8 h in 1 mg/mL DAB.
Real-time RT-PCR
Total RNA from Arabidopsis seedlings were extracted by
the TRIzol method. Reverse transcription was carried out
using 5 μg of total RNA and SuperScript II reverse tran-
scriptase (RT). The 10-fold diluted cDNA was then used as
a template for real-time PCR amplification with the follow-
ing primer pairs: forward primer 5 -AGGAGCCAATCACAGCC-
3 and reverse primer 5 -TCAAGACCAAGCGACCA-3 for
CAT1, forward primer 5 -AACTCCGCCTGCTGTCTG-3 and
reverse primer 5 -ATAGGGCATCAATCCATC-3 for CAT2,
forward primer 5 -TCACAGCCACGCCACTAA-3 and reverse
primer 5 -AGAACCAAGCGACCAACC-3 for CAT3. Real-
time PCR was carried out with the Stratagene (La Jolla,
CA, USA) Mx3005 QPCR systems using SYBR to mon-
itor double-stranded DNA products. For a standard con-
trol, expression of UBQ10 was used with the primers
UBQ10L 5 -ATTACCCGATGGGCAAGTCA-3 and UBQ10R 5 -
CACAAACGAGGGCTGGAACA-3 . The mean value of the
three replicates was normalized using UBQ10 as the internal
control. The relative expression level was then normalized to
the wild type without any treatment.
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(Handling editor: Zhizhong Gong)