Professional Documents
Culture Documents
Catalase Genes Research Paper
Catalase Genes Research Paper
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 biotic/abiotic stress responses (Dat et al. 2000; Apel & Hirt
2004). Photorespiratory H2O2, which is generated during
Photorespiration-associated production of H2O2 accounts
photorespiration in peroxisomes, is the primary source of
for the majority of total H2O2 in leaves of C3 plants and is
H2O2 in C3 plants (Noctor et al. 2002; Karpinski et al. 2003).
mainly eliminated by catalases. In Arabidopsis, lack of Although antioxidative enzymes such as superoxide dismu-
CAT2, but not CAT1 or CAT3, results in growth suppres- tase (SOD) and ascorbate peroxidases (APX) are active
sion and a marked accumulation of H2O2 in leaves. To in leaf peroxisomes, catalase is the foremost scavenging
evaluate the contribution of individual catalase genes and
enzyme for the degradation of photorespiratory H2O2
their promoters to catalase function, we investigated the (Kendall et al. 1983;Willekens et al. 1997; Corpas, Barroso &
growth suppression and H2O2 accumulation phenotypes of del Río 2001; Vandenabeele et al. 2004). Catalase is a tet-
Arabidopsis derivatives expressing catalase genes from het-
rameric iron porphyrin that catalyses the dismutation of
erologous CAT promoters in a cat2 mutant background.
H2O2 to water and oxygen. While low levels H2O2 are elimi-
The expression of CAT2 from the CAT2 promoter restored nated by APX and other peroxidases with the aid of various
the wild-type phenotype in a cat2-1 mutant, while CAT1 reductants like ascorbate and glutathione, catalase, which
and CAT3 promoter-driven expression of CAT2 did not. degrades H2O2 without any reducing power, is mainly active
Ectopic expression of CAT3 from the CAT2 promoter also at relatively high H2O2 concentrations (Gechev et al. 2006).
restored the normal phenotype, unlike that of CAT1 which With the exception of CAT-3 from maize, which is mito-
required replacement of the CAT1 3⬘-untranslated region chondrial (Scandalios et al. 1980), all plant catalases studied
(UTR) with that of CAT2. These results demonstrated that to date have been shown to localize to the peroxisome
the photorespiratory role of CAT2 is determined mainly by (Scandalios 1990; Kamada et al. 2003). Thus, catalase plays a
the regulation of its promoter activity. The 3⬘-UTR of CAT2 principle role in breaking down H2O2 accumulated in per-
was vital for controlling CAT2 protein levels under photo- oxisomes (Gillham & Dodge 1986).
respiratory conditions. Identification of component of There is growing evidence that catalase plays multiple
heterotetramers catalase isoforms suggested that there is roles in a variety of plant tissues at different developmental
some functional redundancy between CAT2 and CAT1 and stages, and it has been implicated in the signal transduction
CAT3. pathways of various stress responses (Fukamatsu, Yabe &
Hasunuma 2003; Verslues et al. 2007). Mutant and trans-
Key-words: 3′-untranslated region; Arabidopsis; catalase; genic plants with reduced catalase expression exhibit
hydrogen peroxide; leaf; photorespiration; promoter- necrotic lesions, are sensitive to oxidative and salt stress,
exchange experiments. 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.
INTRODUCTION 1997; Vandenabeele et al. 2004; Vanderauwera et al. 2005;
Queval et al. 2007). A recent study of the effect of catalase
H2O2 is one of the major reactive oxygen species (ROS), deficiency on ion homeostasis revealed a cross-talk between
and plays an important role in plant biological processes oxidative stress and ethylene (Bueso et al. 2007).
such as seed germination, root elongation (Kwak et al. Like all higher plants, the Arabidopsis genome contains
2003), auxin-regulated root gravitropism (Joo, Bae & three catalase genes (At1g20630, At4g35090 and
Lee 2001), stomatal closure (Murata et al. 2001) and At1g20620), which encode three individual subunits that
Correspondence: Y. T. Lu. Fax: +86 027 68756380; e-mail: yingtlu@ associate to form at least six catalase isoforms (Frugoli et al.
whu.edu.cn 1996). Although the nucleotide and corresponding amino
1656 © 2010 Blackwell Publishing Ltd
Characterization of Arabidopsis catalase genes 1657
acid sequences of the three genes are highly conserved instructions.After treatment of 1 mg of total RNA with RQ1
(Frugoli et al. 1996; McClung 1997), their spatial and tem- RNase-free DNase (Promega, Madison, WI, USA), first-
poral expression patterns are quite different. For example, strand cDNA synthesis was carried out using M-MLV
all three genes are highly expressed in inflorescences, but reverse transcriptase (Promega) according to the manufac-
only CAT2 and CAT3 are highly expressed in leaves under turer’s instructions.
the control of a circadian clock (Frugoli et al. 1996; Zhong For semi-quantitative PCR analysis, 0.5 mL of the 20 mL
et al. 1997). Further analysis of promoter::b-glucuronidase reverse transcription reaction was subjected to PCR (final
(GUS) fusions in transgenic plants showed that CAT2 and volume of 30 mL) using gene-specific primers.As an internal
CAT3 are expressed in different areas in leaf tissues (Zim- control, we analysed ubiquitin, which is constitutively
mermann et al. 2006). CAT2 expression is observed in pho- expressed. Each PCR assay was carried out independently
tosynthetically active tissues and is down-regulated during at least three times. The genes analysed and the correspond-
leaf senescence, whereas CAT3 expression is induced with ing specific primers are listed in Supporting Information
age and corresponds to accumulated H2O2 in vascular Table S1.
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- Detection of H2O2 in Arabidopsis leaves
ing of ROS signalling, because its expression and activity
Plants were grown in soil for 3 weeks and leaves were
are activated by abiotic stresses (Du et al. 2008). Recent
excised. H2O2 contents were determined by the POD-
studies have reported that cat2 mutants exhibit suppressed
coupled assay protocols described by Veljovic-Jovanovic
growth under normal growth conditions and have a defec-
et al. (2001). About 0.1 g Arabidopsis leaves were ground in
tive photorespiration phenotype (Bueso et al. 2007; Queval
liquid N2 and the powder was extracted in 2 mL 1 M HClO4
et al. 2007). Although the expression patterns of catalase
in the presence of insoluble polyvinylpyrrolidone (5%).The
genes have been explored, the contribution of their expres-
homogenate was centrifuged at 12 000 g for 10 min, and the
sion patterns to catalase function is still unclear. Moreover,
supernatant was neutralized with 5 M K2CO3 to pH 5.6 in
data on the specificity and molecular properties of the three
the presence of 100 mL 0.3 M phosphate buffer, pH 5.6. The
catalase gene products are scarce.
solution was centrifuged at 12 000 g for 1 min, and the
Here, we identified and characterized T-DNA insertion
sample was incubated for 10 min with one unit ascorbate
mutants of catalase gene family members. Only cat2
oxidase to oxidize ascorbate prior to assay. The reaction
mutants exhibited significant growth retardation and dis-
mixture was composed of 0.1 M phosphate buffer, pH 6.5,
tinct accumulation of H2O2 in leaves. To gain further insight
3.3 mM 3-(dimethylamino) benzoic acid, 0.07 mM
into the functions of the three catalase genes, we examined
3-methyl-2-benzothiazoline hydrazone and 0.3 units peroxi-
the spatial and temporal expression patterns of the catalase
dase. The reaction was initiated by addition of 200 mL
genes, and the effects of catalase expression from heterolo-
sample. The absorbance change at 590 nm was monitored
gous CAT promoters on the cat2 mutant phenotype.We also
at 25 °C.
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 Generation of transgenic plants for
the three catalase gene products, and that the photorespi-
promoter-GUS analysis, promoter- exchange
ratory role of CAT2 is determined mainly through the regu-
experiments and 3⬘-UTR-exchange experiments
lation of promoter activity and the 3′-untranslated region.
Promoter and genomic fragments of catalase genes were
amplified from genomic DNA of A. thaliana ecotype Col-0
MATERIALS AND METHODS using the appropriate primers (Supporting Information
Plant materials and growth conditions Table S1), and error-free subcloned PCR products were
confirmed by sequencing. For promoter analysis, each pro-
The mutant Arabidopsis lines cat1-1 (CS822253), cat2-1 moter fragment was cloned into the binary vector pBI101.2,
(salk_076998) and cat3-1 (salk_092911) were obtained from which carries GUS and a selectable marker gene for kana-
the Arabidopsis Biological Resource Centre (Columbus, mycin resistance. For promoter-exchange experiments, the
OH, USA). Plants were sown in vermiculite in pots promoter fragments of CAT1 and CAT2 were subcloned
under controlled environmental conditions (23 ⫾ 2 °C, into the BamHI site of the binary vector pCAMBIA1301,
200 mmol m-2 s-1, and a 16 h photoperiod) and given irri- while that of CAT3 was subcloned into the SmaI-PstI sites.
gated nutrient solution three times per week. 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,
Total RNA extraction and semi-quantitative
respectively, and the genomic fragment of CAT2 was cloned
RT-PCR
into the BamHI site of the vector containing CAT1 pro-
Total RNA was isolated using the TRIzol reagent (Invitro- moter and the PstI site of the vector containing CAT3
gen, Carlsbad, CA, USA) according to the manufacturer’s promoter to generate pCAT1::CAT2 and pCAT3::CAT2,
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
1658 Y-Q. Hu et al.
respectively. For the 3′-UTR-exchange experiments, each TBS-T for 10 min each wash. The membrane was incubated
catalase genomic fragment and corresponding 3′-UTR was with anti-AtCAT2 polyclonal antibody (1:500) diluted in
amplified separately. We then used both genomic fragment 1% BSA in TBS-T for 2 h at room temperature. The mem-
and the 3′-UTR as templates to amplify the corresponding brane was washed in TBS-T three times for 10 min each,
chimeric gene, as illustrated in Fig. S1. For example, to and then incubated for 1 h with goat anti-rabbit IgG (1:5000
construct CAT1–CAT2, the CAT1 genomic fragment and dilution in TBS-T) conjugated to horseradish peroxidase.
3′-UTR of CAT2 were used as templates. Chimeric genes Following three washes for 10 min each in TBS-T, immu-
were cloned into pCAMBIA1301, which contained the noreactive proteins were detected using a DAB substrate
CAT2 promoter. The junction sites of all subcloned frag- solution.
ments were confirmed by sequencing. Plasmid constructs
were introduced into Arabidopsis by Agrobacterium tume-
Gel analysis and activity assays of
faciens (GV3101)-mediated transformation using the floral
dip method (Clough & Bent 1984). After selection of trans-
catalase isoforms
formed plants on medium containing 50 mg L-1 kanamycin Tissue from various organs was harvested and frozen at
or 50 mg L-1 hygromycin, the presence of each transgene -80 °C until use. Because CAT2 and CAT3 gene expres-
was verified by PCR using a gene-specific forward primer sion is regulated by circadian rhythm, tissues were always
and a vector-specific reverse primer (see Supporting harvested 6 h after the beginning of illumination. Tissues
Information Table S1). were ground to a powder in liquid nitrogen and then
homogenized in a solution of 100 mM Tris-HCl, pH 8.0,
Antibody production 20% glycerol and 30 mM dithiothreitol (DTT) at 4 °C.
After centrifugation at 14 000 g for 30 min at 4 °C, the
CAT2 was amplified by PCR and subcloned into the supernatant was recovered and protein concentration was
EcoRI site of pET28a for expression in Escherichia quantified according to the method of Bradford (1976)
coli BL21 (DE3). The primers used for PCR were using bovine serum albumin (BSA) as the standard.
pET28aCAT2F and pET28aCAT2R (see Supporting Protein extracts containing 20 mg of total protein were
Information Table S1). All procedures for protein expres- separated by 7.5% native polyacrylamide gel electro-
sion and purification were carried out as previously phoresis with a 3.5% stacking gel for 14 h (70 V) at 4 °C in
described (Li, Hua & Lu 2006). The peptide (CLGQKLA- electrophoresis buffer (250 mM glycine, 25 mM Tris-HCl,
TRLNVRPNF) specific for CAT1 protein was synthesized pH 8.3). Subsequently, the gels were stained for catalase
by the Fmoc (9-fluorenylmethyloxycarbonyl) method activity according to the method of Clare et al. (1984).
(Atherton & Sheppard 1989). Catalase activity assays were performed according to
A rabbit (New Zealand white rabbit) polyclonal antise- Veljovic-Jovanovic et al. (2001).
rum against recombinant AtCAT2 was prepared by subcu-
taneous administration of four injections of 300 mg of
purified recombinant AtCAT2. For the first injection, GUS staining
protein was mixed with 0.15 mL of complete Freund’s GUS staining was carried out according to the methods
adjuvant plus 0.11 mL of incomplete adjuvant. The anti- of Jefferson (1987). Briefly, tissues or whole plants
peptide antibody was produced from a rabbit by injecting were incubated at 37 °C overnight in staining solution
with the synthetic peptide conjugated to BSA, which (100 mM sodium phosphate buffer, pH 7.5, containing
had been activated by MBS (m-maleimidobenzoly-N- 10.0 mM EDTA, pH 8.0, 0.5 mM K3[Fe(CN)6] and
hydroxysuccinimide ester). Subsequent booster injections 0.5 mM K4[Fe(CN)6], 0.1% Triton X-100 and 1.0 mM
were administered at approximately 2 week intervals, and 5-bromo–chloro-3-indolyl-b-D-glucuronide). Chlorophyll
contained protein mixed (1:1) with incomplete adjuvant. was removed by rinsing the plant material in 70% ethanol.
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 RESULTS
at -80 °C. Genomic characterization of cat1-1, cat2-1,
cat3-1, and cat2-1/cat3-1 insertion mutants
Western blot analysis of Arabidopsis
Tissue extracts containing 20 mg of total protein were mixed To obtain knockout mutants of the three Arabidopsis cata-
with 2 mL of 5XSDS-PAGE sample buffer (Sambrook et al. lase genes, we selected T-DNA insertion lines from the
1989) and then heated for 3 min at 95 °C. Proteins were Arabidopsis Biological Resource Center (ABRC) (Fig. 1a).
separated by 10% SDS-polyacrylamide gel electrophoresis Using PCR with specific primers to analyse plants grown
and then electroblotted onto a nitrocellulose membrane. from T3 seeds, we identified homozygous T-DNA insertion
The membrane was blocked in 5% non-fat dry milk in lines (Fig. 1b), which we designated cat1-1, cat2-1 and
TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% cat3-1. T4 seeds obtained from the identified homozygotes
Tween 20) overnight at 4 °C and then washed three times in were used for all further analyses. Because CAT2 and CAT3
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
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- approximately 33% that of wild-type fresh weight (Fig. 2c).
erated crosses of cat2-1 and cat3-1 to obtain a homozygous The cat2-1/cat3-1 double mutant exhibited a slightly more
double insertion mutant, which was also confirmed by PCR severe phenotype than the cat2-1 mutant (Fig 2–c). These
(Fig. 1b). The resulting double mutant was designated cat2- observations were consistent with previous reports by
1/cat3-1. When we analysed catalase gene expression in others (Bueso et al. 2007; Queval et al. 2007). Compared to
each of the cat mutants using RT-PCR and gene-specific wild-type plants, cat1-1, cat2-1, cat3-1, and cat2-1/cat3-1
primers that targeted the 3′-UTRs of the genes, we were exhibited variable reductions in catalase activity, retaining
unable to detect corresponding transcripts in any of the 92%, 24%, 83% and 7% of total wild type catalase activity,
mutants (Fig. 1c), indicating that they were complete gene respectively (Fig. 2d).
knockouts.
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, Expression of catalase genes under the control
we analysed longer promoter sequences, consisting of over of heterologous CAT promoters differentially
2000 basepairs upstream of the cat start codon. As the affects the phenotype the cat2-1 mutant
abnormal phenotype of the cat2 mutant predominantly
affected leaves, we focused on GUS activity in the leaves of The expression patterns of the three catalase genes in
transgenic plants. The expression of CAT1 was abundant in leaves are quite different. To examine the role of differen-
the cotyledon of early seedlings (Fig. 3a), and decreased tial catalase gene expression on catalase function, we placed
with development (Fig. 3b). CAT1 expression was nearly expression of the catalase genes under the control of het-
undetectable in young or mature rosette leaves (Fig 3c & e), erologous CAT promoters, and examined the effect on the
but was induced and began to increase upon senescence cat2-1 mutant phenotype. Expression of a genomic frag-
(Fig. 3d). The expression of CAT2 localized predominantly ment incorporating the CAT2 locus under the control of the
to the mesophyll cells of green leaves (Fig 3f–h & j). As CAT2 promoter was able to complement the cat2-1 mutant
yellow cotyledons turned green, CAT2 expression levels phenotype; all 45 cat2-1/pCAT2::CAT2 transgenic plants
gradually rose (Fig 3f & g). CAT2 expression was down- were restored to the wild-type phenotype (Fig 4a & c–e).
regulated during leaf senescence and was much less abun- We then generated the following chimeric genes by placing
dant in senescent leaves (Fig. 3i). CAT3 was highly catalase coding regions, including part of the 3′-downstream
expressed in the vasculature of senescent cotyledon and region, under the control of a heterologous CAT promoter,
rosette leaves, but less so in mesophyll cells adjacent to the resulting in pCAT1::CAT2, pCAT3::CAT2, pCAT2::CAT3
vasculature (Fig 3l, m & o). CAT3 was weakly expressed in and pCAT2::CAT1. The chimeric genes were introduced
senescent leaves (Fig. 3n). together with the hygromycin resistance marker into a
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
Characterization of Arabidopsis catalase genes 1661
(c) (d)
(h) (i)
(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
plants, which expressed a chimeric CAT1 coding region and there were high levels of photorespiratory H2O2 (Fig. 4c).
CAT2 3′-UTR, 39 exhibited full complementation, in that Although the expression of CAT3 was abundant in mature
the levels of H2O2 in leaves and rosette fresh weight were leaves, expression was specific to vascular tissues (Fig. 3).
similar to wild-type plants (Fig 7b & c), and catalase activity Further analysis revealed that in cat2-1/pCAT3::CAT2
in leaves was restored to 88% of wild-type levels (Fig. 7d). transgenic plants (CAT2 under control of the CAT3 pro-
By comparison, there was a much higher accumulation of moter), CAT2-specific isoform activity in CAT3-expressing
H2O2 in leaves of cat2-1/pCAT2::CAT2-CAT1 transgenic regions was readily detected (Fig. 6b). However, while total
plants as compared to cat2-1/pCAT2::CAT2 plants (Fig. 7b), catalase activity was increased by approximately 50%
and the rosette fresh weight and catalase activity in leaves (Fig. 4e), the accumulation of H2O2 in leaves was not mark-
reverted to 50% and 48% of wild type, respectively (Fig 7c edly decreased (Fig. 4c). In conclusion, the results clearly
& d). Biochemical analysis of catalase proteins showed that demonstrated that the photorespiratory role of CAT2
CAT1 catalase activity was increased in leaves of cat2-1/ is determined mainly by the regulation of its promoter
pCAT2::CAT1-CAT2 plants, whereas catalase activity con- activity
tributed by CAT2 in leaves of cat2-1/pCAT2::CAT2-CAT1 The 3′-UTR is crucial for several aspects of post-
plants was decreased significantly as compared to cat2-1/ transcriptional regulation, including mRNA transport from
pCAT2::CAT2 transgenics (Fig 8a & b). the nucleus into the cytoplasm, enhancement of mRNA
transcript stability and regulation of mRNA translation
(Zhao, Hyman & Moore 1999; Grzybowska, Wilczynska &
DISCUSSION
Siedlecki 2001; Proudfoot, Furger & Dye 2002). The
Based on molecular analyses of catalase gene expression in absence of a 3′-UTR has been shown to result in low trans-
different plants (Ni & Trelease 1991; Willekens et al. 1994; lational efficiency of cat1 mRNA in rye leaves in vitro
Suzuki et al. 1995), plant catalases have been subdivided (Schmidt, Grief & Feierabend 2006). In the current study,
into three classes (Willekens, Inzé & Van Montagu 1995). we investigated the function of the 3′-UTR of Arabidopsis
Unlike Class III catalases, which are highly expressed in CAT2 mRNA in vivo. When driven by the same CAT2
young seedlings and senescent tissues, Class I and Class II promoter, the replacement of the CAT2 3′-UTR by that of
catalases are abundant in mature plants. While Class II CAT1 led to obvious decreases in levels of CAT2 subunit as
catalases are mainly expressed in vascular tissues, Class I well as catalase activity contributed by CAT2 (Fig. 8), and
catalases are light-dependent and are most prominent in accompanied by a marked increase in the levels of H2O2 in
photosynthetic cells. Thus, it has been postulated that Class leaves (Fig. 7b). On the other hand, when driven by the
I catalases are more important in the removal of H2O2 CAT2 promoter, transcription of CAT1 in leaves increased
produced during photorespiration than members of the significantly, but there were no obvious changes detected in
other classes (Willekens et al. 1994, 1995; Zimmermann the levels of CAT1 subunit. When the 3′-UTR of CAT1 was
et al. 2006). Recent studies of an Arabidopsis cat2 mutant replaced by that of CAT2, active CAT1 was expressed
have verified this hypothesis (Bueso et al. 2007; Queval et al. under the control of the CAT2 promoter and photorespira-
2007). The results of the current study confirm and expand tory H2O2 was efficiently eliminated (Fig. 8). These results
these earlier findings. Firstly, a major photorespiratory role suggested that the 3′-UTR of CAT2 is vital for regulating
of CAT2 was demonstrated by the finding that the loss of the protein level of CAT2 under photorespiratory
neither CAT1 nor CAT3 resulted in a photorespiration phe- conditions.
notype, and the loss of CAT3 did not markedly aggravate There is growing evidence that H2O2 serves as a major
the phenotype of the cat2 mutant (Fig. 2). Secondly, an signalling molecule in various aspects of cellular function in
important regulatory role of the CAT2 promoter was plants (Apel & Hirt 2004; Foyer & Noctor 2005; Vanderau-
demonstrated by complementation experiments, in which wera et al. 2005; Gechev et al. 2006). However, our under-
expression of CAT2 and ectopic expression of CAT3 or the standing of the mechanisms that control H2O2 production
chimeric gene CAT1-CAT2 complemented the cat2 mutant and metabolism is relatively limited. As a scavenger or
phenotype when they were placed under the control of the door-keeper for H2O2, catalase plays an important role not
CAT2 promoter (Figs 4 and 7). Our results suggest that the only in protecting cells against oxidant stress, but also in
mechanism by which CAT1 and CAT3 are restricted from governing cellular redox homeostasis and by extension, the
participating in the photorespiratory process involves regu- regulation of cellular signalling (Baker & Orlandi 1995;
lation at the level of expression. Promoter-GUS analysis Xing, Jia & Zhang 2008; Foyer et al. 2009). Photorespiration
revealed that CAT1, a Class III catalase, is expressed mainly is by far the fastest H2O2-producing system in photosyn-
in senescent leaves (Fig. 3). In agreement with this, in cat2- thetic cells (Noctor et al. 2002). There is good evidence that
1/pCAT1::CAT2 transgenic plants, which expressed CAT2 reduced catalase expression under photorespiratory condi-
under the control of the CAT1 promoter, CAT2 isoform tions causes dramatic adjustments in the glutathione pool
activity in CAT1-expressing regions was detected only in (Smith et al. 1985; Queval et al. 2007, 2009), a key thiol/
senescent leaves (50–75% loss of chlorophyll) (Fig. 6c), in disulfide buffer in cellular redox homeostasis (Foyer &
which degradation of chlorophylls and Rubisco leads to a Noctor 2005). On the other hand, H2O2 production and
rapid decline in photosynthetic activity and photorespira- signalling under abiotic stresses could also activate the
tion (Friedrich & Huffaker 1980; Jiang et al. 1993), and expression and activity of catalase (Orendi et al. 2001; Xing
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
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
Characterization of Arabidopsis catalase genes 1667
CAT3, while the localization of CAT1 and CAT3 is distinct. species during plant stress responses. Cellular and Molecular
CAT1, a Class III catalase, is predicted to localize to gly- Life Sciences 57, 779–795.
oxysomes (Willekens et al. 1995). Glyoxysomes function in Du Y.Y., Wang P.C., Chen J. & Song C.P. (2008) Comprehensive
functional analysis of the catalase gene family in Arabidopsis
the conversion of lipids into sucrose and are present mainly
thaliana. Journal of Integrative Plant Biology 50, 1318–1326.
in storage organs such as endosperms and cotyledons Feierabend J. (2005) Catalases in plants: molecular and functional
during post-germinative growth in oil-seed plants, as well as properties role in stress defence. In Antioxidants and Reactive
in senescent organs (Beevers 1982; Nishimura et al. 1996). Oxygen Species in Plants (ed. N. Smirnoff), pp. 101–140. Black-
In these tissues, catalase isoforms composed of CAT1 and well Publishing, Oxford.
CAT2 are readily detected (Zimmermann et al. 2006 and Fita I. & Rossmann M.G. (1985) The active center of catalase.
Journal of Molecular Biology 185, 21–37.
Fig. 6c). The expression of CAT2 in young cotyledons and
Foyer C.H. & Noctor G. (2005) Redox homeostasis and antioxidant
senescent leaves (Fig 3f & i) raises the interesting possibil- signaling: a metabolic interface between stress perception and
ity that CAT2 may also play a role in glyoxysomal removal, physiological responses. The Plant Cell 17, 1866–1875.
as well as CAT1. On the other hand, while CAT3, a Class II Foyer C.H., Bloom A.J., Queval G. & Noctor G. (2009) Photores-
catalase, was predominantly expressed in vascular tissues, it piratory metabolism: genes, mutants, energetics, and redox
was also expressed in mesophyll cells adjacent to the vas- signaling. Annual Review of Plant Biology 60, 455–484.
culature (Fig 3m & o). Thus, it is possible that CAT3 may be Friedrich J.W. & Huffaker R.C. (1980) Photosynthesis, leaf resis-
tances, and ribulose-1,5-bisphosphate carboxylase degradation
active in these tissues as well as CAT2. These results suggest
in senescing barley leaves. Plant Physiology 65, 1103–1107.
that although CAT2 is more important in the removal of Frugoli J.A., Zhong H.H., Nuccio M.L., McCourt P., McPeek M.A.,
H2O2 produced during photorespiration, there is some func- Thomas T.L. & McClung C.R. (1996) Catalase is encoded by a
tional redundancy with CAT1 and CAT3. Our approach to multigene family in Arabidopsis thaliana (L.) Heynh. Plant
studying the components of catalase isoforms provides a Physiology 112, 327–336.
valuable tool for further research into the functions of the Fukamatsu Y., Yabe N. & Hasunuma K. (2003) Arabidopsis NDK1
catalase family. 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.
ACKNOWLEDGMENTS (2006) Reactive oxygen species as signals that modulate plant
stress responses and programmed cell death. BioEssays: News
This work was supported in part by Major State Basic and Reviews in Molecular, Cellular and Developmental Biology
Research Program (2007CB108701), National Natural 28, 1091–1101.
Science Foundation of China. Gillham D.J. & Dodge A.D. (1986) Hydrogen peroxide-scavenging
systems with pea chloroplasts. A quantitative study. Planta 167,
246–251.
REFERENCES Gouet P., Jouve H.M. & Dideberg O. (1995) Crystal structure of
Proteus mirabilis PR catalase with and without bound NADPH.
Apel K. & Hirt H. (2004) Reactive oxygen species: metabolism, Journal of Molecular Biology 249, 933–954.
oxidative stress, and signal transduction. Annual Review of Plant Grzybowska E.A., Wilczynska A. & Siedlecki J.A. (2001) Regula-
Biology 55, 373–399. tory functions of 3′UTRs. Biochemical and Biophysical Research
Atherton E. & Sheppard R.C. (1989) Solid Phase Peptide Synthesis: Communications 288, 291–295.
A Practical Approach. IRL Press, Oxford. Heinze M. & Gerhardt B. (2002) Plant catalase. In Plant Peroxi-
Baker C.J. & Orlandi E.W. (1995) Active oxygen in plant patho- somes: Biochemistry, Cell Biology and Biology and Biotechno-
genesis. Annual Review of Phytopathology 33, 299–321. logical Application (eds A. Baker & I.A. Graham), pp. 103–140.
Beevers H. (1982) Glyoxysomes in higher plants. Annals of the Kluwer Academic Publisher, Dordrecht.
New York Academy of Sciences 386, 243–253. Jefferson R.A. (1987) Assaying chimeric genes in plants: the GUS
Bradford M.M. (1976) A rapid and sensitive method for the quan- Gene Fusion System. Plant Molecular Biology Reporter 5, 387–
titation of microgram quantities of protein utilizing the principle 405.
of protein-dye binding. Analytical Biochemistry 72, 248–254. Jiang C.Z., Rodermel S.R. & Shibles R.M. (1993) Photosynthesis,
Bueso E., Alejandro S., Carbonell P., Perez-Amador M.A., Fayos J., rubisco activity and amount, and their regulation by transcrip-
Belles J.M., Rodriguez P.L. & Serrano R. (2007) The lithium tion in senescing soybean leaves. Plant Physiology 101, 105–
tolerance of the Arabidopsis cat2 mutant reveals a cross-talk 112.
between oxidative stress and ethylene. The Plant Journal 52, Joo J.H., Bae Y.S. & Lee J.S. (2001) Role of auxin-induced reactive
1052–1065. oxygen species in root gravitropism. Plant Physiology 126, 1055–
Clare D.A., Duong M.N., Darr D., Archibald F. & Fridovich I. 1060.
(1984) Effects of molecular oxygen on detection of superoxide Kamada T., Nito K., Hayashi H., Mano S., Hayashi M. & Nishimura
radical with nitroblue tetrazolium and on activity stains for cata- M. (2003) Functional differentiation of peroxisomes revealed by
lase. Analytical Biochemistry 140, 532–537. expression profiles of peroxisomal genes in Arabidopsis thaliana.
Clough S.J. & Bent A.F. (1984) Floral dip: a simplified method for Plant & Cell Physiology 44, 1275–1289.
Agrobacterium -mediated transformation of Arabidopsis Karpinski S., Gabrys H., Mateo A., Karpinska B. & Mullineaux
thaliana. The Plant Journal 16, 735–743. P.M. (2003) Light perception in plant disease defence signalling.
Corpas F.J., Barroso J.B. & del Río L.A. (2001) Peroxisomes as a Current Opinion in Plant Biology 6, 390–396.
source of reactive oxygen species and nitric oxide signal mol- Kendall A.C., Keys A.J., Turner J.C., Lea P.J. & Miflin B.J. (1983)
ecules in plant cells. Trends in Plant Science 6, 145–150. The isolation and characterisation of a catalase-deficient mutant
Dat J., Vandenabeele S., Vranová E, Van Montagu M., Inzé D. & of barley (Hordeum vulgare L.). Planta 159, 505–511.
Van Breusegem F. (2000) Dual action of the active oxygen Kwak J.M., Mori I.C., Pei Z.M., Leonhardt N., Torres M.A., Dangl
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
Characterization of Arabidopsis catalase genes 1669
J.L., Bloom R.E., Bodde S., Jones J.D. & Schroeder J.I. (2003) Scandalios J.G., Tong W.F. & Roupakias D.G. (1980) Cat3, a third
NADPH oxidase AtrbohD and AtrbohF genes function in ROS- gene locus coding for a tissue-specific catalase in maize: genetics,
dependent ABA signaling in Arabidopsis. The EMBO Journal intracellular location, and some biochemical properties. Molecu-
22, 2623–2633. lar and General Genetics 179, 33–41.
Li R.J., Hua W. & Lu Y.T. (2006) Arabidopsis cytosolic glutamine Scandalios J.G. (1990) Regulation and properties of plant catalase.
synthetase AtGLN1;1 is a potential substrate of AtCRK3 In Causes of Photooxidative Stress and Amelioration of Defense
involved in leaf senescence. Biochemical and Biophysical Systems in Plants (eds C.H. Foyer & P.M. Mullineaux), pp. 275–
Research Communications 342, 119–126. 315. CRC Press, Boca Raton, FL.
Loprasert S., Vattanaviboon P., Praituan W., Chamnongpol S. & Schmidt M., Dehne S. & Feierabend J. (2002) Post-transcriptional
Mongkolsuk S. (1996) Regulation of the oxidative stress protec- mechanisms control catalase synthesis during its light-induced
tive enzymes, catalase and superoxide dismutase in Xanthomo- turnover in rye leaves through the availability of the hemin
nas – a review. Gene 179, 33–37. cofactor and reversible changes of the translation efficiency of
McClung C.R. (1997) Regulation of catalases in Arabidopsis. Free mRNA. Plant Journal 31, 601–613.
Radical Biology & Medicine 23, 489–496. Schmidt M., Grief J. & Feierabend J. (2006) Mode of translational
Michelet L., Zaffagnini M., Marchand C. et al. (2005) Glutathiony- activation of the catalase (cat1) mRNA of rye leaves (Secale
lation of chloroplast thioredoxin f is a redox signaling mecha- cereale L.) and its control through blue light and reactive oxygen.
nism in plants. Proceedings of the National Academy of Sciences Planta 223, 835–846.
102, 16478–16483. Smith I.K., Kendall A.C., Keys A.J., Turner J.C. & Lea P.J. (1985)
Michelet L., Zaffagnini M., Vanacker H., Le Marechal P., Marchand The regulation of the biosynthesis of glutathione in leaves of
C., Schroda M., Lemaire S.D. & Decottignies P. (2008) In vivo barley (Hordeum vulgare L.). Plant Science 41, 11–17.
targets of S-thiolation in Chlamydomonas reinhardtii. Journal of Suzuki M., Miyamoto R., Hattori T., Nakamura K. & Asahi T.
Biological Chemistry 283, 21571–21578. (1995) Differential regulation of the expression in transgenic
Murata Y., Pei Z.M., Mori I.C. & Schroeder J. (2001) Abscisic acid tobacco of the gene for beta-glucuronidase under the control of
activation of plasma membrane Ca(2+) channels in guard cells the 5′-upstream regions of two catalase genes from castor bean.
requires cytosolic NAD(P)H and is differentially disrupted Plant & Cell Physiology 36, 273–279.
upstream and downstream of reactive oxygen species production Tatusova T.A. & Madden T.L. (1999) Blast 2 sequences, a new tool
in abi1-1 and abi2-1 protein phosphatase 2C mutants. The Plant for comparing protein and nucleotide sequences. FEMS Micro-
Cell 13, 2513–2523. biology Letters 174, 247–250.
Ni W. & Trelease R.N. (1991) Post-Transcriptional Regulation of Vanacker H., Carver T.L. & Foyer C.H. (2000) Early H2O2 accu-
Catalase Isozyme Expression in Cotton Seeds. The Plant Cell 3, mulation in mesophyll cells leads to induction of glutathione
737–744. during the hyper-sensitive response in the barley-powdery
Nishimura M., Hayashi M., Kato A., Yamaguchi K. & Mano S. mildew interaction. Plant Physiology 123, 1289–1300.
(1996) Functional transformation of microbodies in higher plant Vandenabeele S., Vanderauwera S., Vuylsteke M., Rombauts S.,
cells. Cell Structure and Function 21, 387–393. Langebartels C., Seidlitz H.K., Zabeau M., Van Montagu M.,
Noctor G., Veljovic-Jovanovic S., Driscoll S., Novitskaya L. & Foyer Inzé D. & Van Breusegem F. (2004) Catalase deficiency drasti-
C.H. (2002) Drought and oxidative load in the leaves of C3 cally affects gene expression induced by high light in Arabidopsis
plants: a predominant role for photorespiration? Annals of thaliana. The Plant Journal 39, 45–58.
Botany 89, 841–850. Vanderauwera S., Zimmermann P., Rombauts S., Vandenabeele S.,
Orendi G., Zimmermann P., Baar C. & Zentgraf U. (2001) Loss of Langebartels C., Gruissem W., Inzé D. & Van Breusegem F.
stress-induced expression of catalase3 during leaf senescence in (2005) Genome-wide analysis of hydrogen peroxide-regulated
Arabidopsis thaliana is restricted to oxidative stress. Plant gene expression in Arabiodopsis reveals a high light-induced
Science 161, 301–314. transcriptional cluster involved in anthocyanin biosynthesis.
Proudfoot N.J., Furger A. & Dye M.J. (2002) Integrating mRNA Plant Physiology 139, 806–821.
processing with transcription. Cell 108, 501–512. Veljovic-Jovanovic S.D., Pignocchi C., Noctor G. & Foyer C.H.
Queval G., Issakidis-Bourguet E., Hoeberichts F.A., Vandorpe M., (2001) Low ascorbic acid in the vtc-1 mutant of Arabidopsis is
Gakiere B., Vanacker H., Miginiac-Maslow M., Van Breusegem associated with decreased growth and intracellular redistri-
F. & Noctor G. (2007) Conditional oxidative stress responses in bution of the antioxidant system. Plant Physiology 127, 426–
the Arabidopsis photorespiratory mutant cat2 demonstrate that 435.
redox state is a key modulator of daylength-dependent gene Verslues P.E., Batelli G., Grillo S., Agius F., Kim Y.S., Zhu J.,
expression, and define photoperiod as a crucial factor in the Agarwal M., Katiyar-Agarwal S. & Zhu J.K. (2007) Interaction
regulation of H2O2-induced cell death. The Plant Journal 52, of SOS2 with nucleoside diphosphate kinase 2 and catalases
640–657. reveals a point of connection between salt stress and H2O2 sig-
Queval G., Thominet D., Vanacker H., Miginiac-Maslow M., naling in Arabidopsis thaliana. Molecular and Cellular Biology
Gakiere B. & Noctor G. (2009) H2O2-activated up-regulation of 27, 7771–7780.
glutathione in Arabidopsis involves induction of genes encoding Willekens H., Langebartels C., Tire C., Van Montagu M., Inzé D. &
enzymes involved in cysteine synthesis in the chloroplast. Van Camp W. (1994) Differential expression of catalase genes in
Molecular Plant 2, 344–356. Nicotiana plumbaginifolia (L.). Proceedings of the National
Sambrook J., Fritsch E.F. & Maniatis T. (1989) Molecular Cloning: Academy of Sciences of the United States of America 91, 10450–
A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory 10454.
Press, Cold Spring Harbor, NY. Willekens H., Inzé D. & Van Montagu M. (1995) Catalases in
Santos I., Pires H., Almeida J.M., Fidalgo F., Confraria A., Duarte plants. Molecular Breeding 1, 207–208.
M., Borlido J. & Salema R. (2006) Phylogenetic relationship of Willekens H., Chamnongpol S., Davey M., Schraudner M.,
potato CAT1 and CAT2 genes, their differential expression in Langebartels C., Van Montagu M., Inze D. & Van Camp W.
non-photosynthetic organs and during leaf development, and (1997) Catalase is a sink for H2O2 and is indispensable for
their association with different cellular processes. Functional stress defence in C3 plants. The EMBO Journal 16, 4806–
Plant Biology 33, 639–651. 4816.
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670
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.
© 2010 Blackwell Publishing Ltd, Plant, Cell and Environment, 33, 1656–1670