ORIGINAL PAPER

Phylogenetic relationships and expression in response to lowtemperature of a catalase gene in banana (Musa acuminatacv. ‘‘Grand Nain’’) fruit

Luis Figueroa-Yanez • Julia Cano-Sosa • Enrique Castano • Ana-Ly Arroyo-Herrera •

Jose Humberto Caamal-Velazquez • Felipe Sanchez-Teyer • Rodolfo Lopez-Gomez •

Cesar De Los Santos-Briones • Luis Rodrıguez-Zapata

Received: 19 August 2011 / Accepted: 23 December 2011 / Published online: 7 January 2012

� Springer Science+Business Media B.V. 2012

Abstract For many plants, particularly those of tropical

and subtropical origin, chilling injury occurs as a result of

their exposure to low, but nonfreezing temperatures. Banana

fruits are highly susceptible to chilling injury, but little is

known about the role of genes that scavenge reactive oxygen

species in fruits during chilling injury. In this study, a cata-

lase gene, designated MaCat2, was isolated from Musa

acuminata cv. Grand Nain fruits. The full-length cDNA

sequence is 1,479 bp, and based on phylogenetic analysis, it

is related to catalase type 2 genes from Elaeis guineensis and

Zantedeschia aethiopica. Expression studies revealed that

the MaCat2 gene was induced in severe stress of banana

fruits. MaCat2 expression in banana peel increased in

response to both low temperature and physical damage, but

not so under heat stress or during normal fruit ripening. These

findings suggest that MaCat2 is induced in banana peel by

cold treatment and is regulated at transcriptional level, pos-

sibly playing a role in chilling injury response of banana fruit.

Keywords Abiotic stress � Chilling injury �Gene expression � Hydrogen peroxide

Introduction

Bananas and plantains (except for Fe’i bananas) are derived

from Musa acuminata Colla, or from hybrids of this species

with M. balbisiana Colla (Gawel and Jarret 1991). Musa

species grow in the tropical and subtropical world regions

and have varied human uses, ranging from the edible

bananas and plantains of the tropics to cold-hardy fiber and

ornamental plants. Bananas are originated from tropical

regions and several abiotic stresses affect their growth

(Zhang et al. 2011). Chilling injury (CI) is one of the prin-

cipal natural disasters occurring in the majority of the

banana producing regions. CI refers to the physiological

damage that occurs in plants and plant products as a result of

their exposure to low but nonfreezing temperatures. The

substantial economic consequences of CI have provided the

incentive for studying effective ways to alleviate the mani-

fest symptoms of this disorder. Plants display a variety of

responses to low temperature (LT) stress, including altera-

tions in ethylene biosynthesis, increased respiration rates,

cessation of protoplasmic streaming, increased solute leak-

age and uncoupling of oxidative phosphorylation. These

various responses ultimately give rise to an array of visual

symptoms, which can render severe losses in product qual-

ity both pre- and postharvest. Skin darkening during cold

L. Figueroa-Yanez � A.-L. Arroyo-Herrera � F. Sanchez-Teyer �C. De Los Santos-Briones � L. Rodrıguez-Zapata (&)

Unidad de Biotecnologıa, Centro de Investigacion Cientıfica de

Yucatan, A.C. Calle 43 No. 130 Col. Chuburna de Hidalgo, 97200

Merida, Yucatan, Mexico

e-mail: lcrz@cicy.mx

J. Cano-Sosa � E. Castano

Unidad de Bioquımica y Biologıa Molecular de Plantas, Centro

de Investigacion Cientıfica de Yucatan, A.C. Calle 43 No. 130

Col. Chuburna de Hidalgo, 97200 Merida, Yucatan, Mexico

J. Cano-Sosa

Centro de Investigacion y Asistencia en Tecnologıa y Diseno del

Estado de Jalisco, A.C. Calle 30, No 151. Interior Canacintra,

Garcıa Gineres, 97070 Merida, Yucatan, Mexico

J. H. Caamal-Velazquez

Colegio de Postgraduados campus Campeche, Nicaragua

No 91 3er piso, 24050 Campeche, Campeche, Mexico

R. Lopez-Gomez

Instituto de Investigaciones Quımico-Biologicas,

Universidad Michoacana de San Nicolas de Hidalgo,

58060 Morelia, Michoacan, Mexico

123

Plant Cell Tiss Organ Cult (2012) 109:429–438

DOI 10.1007/s11240-011-0107-4

storage is a common CI symptom in banana fruit (Kim and

Lee 1988). Recent results indicate that the plantain cultivar

‘Cachaco’ (Musa paradisiaca ABB cv. ‘Dajiao’) exhibits

higher activity of free radical scavenging enzymes at low

temperature as compared to the banana cultivar ‘Williams’

(Musa acuminata AAA cv. ‘Williams’), which to a certain

extent can explain the higher cold tolerance of the plantain.

They found that exposure to LT resulted in an increased

content of malondialdehyde (MDA), hydrogen peroxide

(H2O2) and superoxide radical (O2� -), and a decrease of

photochemical efficiency (Fv/Fm) and net photosynthetic

rate (PN), but the cultivar ‘Cachaco’ showed a better LT

tolerance than the cultivar ‘Williams.’ After LT treatment,

total free radical scavenging capability (DPPH� scavenging

capability) showed a significant decrease in ‘Williams’ but

no significant alternations were found in ‘Cachaco.’ After

LT treatment, ‘Cachaco’ displayed a significant increase in

the activity of ascorbate peroxidase (APX) and peroxidase

(POD), superoxide dismutase (SOD) showed no significant

response and catalase (CAT) showed a significantly

decreased activity. However, the activities of the four above-

mentioned antioxidant enzymes showed a significant

decrease in seedlings of ‘Williams’ exposed to LT (Zhang

et al. 2011). Catalase (CAT; H2O2:H2O2 oxidoreductase, EC

1.11.1.6) is an enzyme that, together with superoxide dis-

mutases and hydroperoxidases, makes up a defense system

for the scavenging of superoxide radicals and hydroperox-

ides (Beyer and Fridovich 1987; Williamson and Scandalios

1992). Catalases in plants are differentially regulated with

regard to the type of tissue or developmental stage, as well as

by a variety of environmental stresses (Esaka et al. 1997;

McClung 1997; Scandalios et al. 1997; Willekens et al.

1994a, b; Yi et al. 2003). The presence of multiple isoforms

of the enzyme, and their complex regulation, suggests that

each isoform plays a different role. The catalase isoforms

encoded by the Nicotiana plumbaginifolia CAT1 gene are

thought to play a photorespiratory role in leaves (Willekens

et al. 1994a), and a similar role has been suggested for iso-

forms of the Arabidopsis thaliana CAT2 gene, both genes

having similar expression (Frugoli et al. 1996). Two Nico-

tiana tabacum catalases are regulated by salicylic acid (SA),

a well-known regulator of catalase gene expression. Pres-

ence of a SA sensitive catalase is also indicative of its

involvement in systemic acquired resistance to pathogens

(Chen et al. 1993). In Capsicum annuum and Triticum aes-

tivum there is evidence that Cat1 and Cat2 are expressed

differently during the circadian rhythm or under severe

drought treatment (Lee and An 2005; Luna et al. 2005). A

significant number of catalase sequences have been repor-

ted. Guan and Scandalios (1996) described a phylogenetic

reconstruction of 16 plant catalase sequences and discussed

possible evolutionary relationships to bacterial and other

eukaryotic catalases. In addition, Mayfield and Duvall

(1996) described anomalous groupings of catalase gene

sequences using a subset of nine bacterial and five eukary-

otic sequences. Both of these reports utilized small subsets

of the total number of catalase sequences available. Che-

likani et al. (2004) analyzed 300 catalase sequences from

different prokaryotic and eukaryotic organisms, among

which approximately 225 are monofunctional, 50 are

bifunctional with a heme binding site and 25 contain a

magnesium binding site. Willekens et al. (1994a) reported

17 catalase cDNAs from different plants, also showing that

catalases of the same type can play different specific roles.

However, until now there is little information about which

catalase genes are expressed during low temperature in

tropical fruits, a knowledge that would, first, be of aid as a

marker as is the case of some catalases in different types of

stress, and second, would be useful in subsequent efforts for

trying to understand the specific function of such expressed

genes. In the present paper we provide the complete

sequence of a catalase type 2 gene from banana (MaCat2)

together with its expression profile in fruit during LT treat-

ment, and we show its phylogenetic relationship with the 14

known catalases of monocot plants reported up to date.

Materials and methods

Stress induction treatments

Banana fruits were obtained from Musa acuminata cv.

‘Grand Nain’ plants grown at 25�C in a greenhouse. Fruits

were harvested at ripening stage number 4, according to the

peel color stages proposed by the Customers Services

Department of Chiquita Brands, Inc., Cincinnati, OH. Fruits

were treated by wounding, exposure to high or to low tem-

perature and allowed to mature naturally. Wounding lesions

were generated with forceps by hitting the fruits two to three

times. For high or low temperature treatments, banana fruits

were incubated for 8 h in a diurnal growth chamber (Forma

Scientific) at 25, 10 or 45�C (Caamal-Velazquez et al. 2007).

In the case of maturation, all samples were taken from a

single hand (i.e. a pair of two adjacent rows of fruits) in order

to avoid heterogeneity due to differences in development.

The weight of the pulp and peel was determined separately

and material was deep-frozen at -80�C. Vegetative tissue

(leaf, pseudostem, corm and root) was obtained from

6-month old banana plants that were incubated for 24 h at

10�C and the collected material was frozen at -80�C.

Total RNA extraction

RNA was obtained according to the protocol of Medina-

Escobar et al. (1997). Briefly, 2 g of fruit tissue were frozen

in liquid nitrogen and ground. The ground material was

430 Plant Cell Tiss Organ Cult (2012) 109:429–438

123

homogenized in 10 mL RNA extraction buffer (100 mM

Tris–HCl pH 8, 50 mM EDTA, 500 mM NaCl, 1.4%

b-mercaptoethanol and 2% SDS) and incubated for 15 min

at 65�C. After incubation, 1.5 M potassium acetate was added

to the mixture, and samples were incubated for 10 min at

-20�C. The supernatant was obtained by centrifugation

(17,0009g for 30 min) at 4�C. The aqueous phase was

recovered, and the total RNA was precipitated by addition of

one volume of isopropanol. Samples were incubated for 1 h at

-20�C and centrifuged for 30 min at 4�C. Pellets were

resuspended in TE (100 mM Tris–HCl pH 8, 50 mM EDTA),

after which 300 mM acetate sodium and 50% isopropanol

were added. Samples were incubated overnight at -20�C and

centrifuged (17,0009g for 30 m) at 4�C. Pellets were washed

with 70% EtOH, centrifuged, resuspended in nuclease free-

water and stored at -80�C until use.

Isolation of full length MaCat2 transcript

Differential display was used to identify the MaCat2 partial

sequence gene of banana fruit in response to low temperature

stress (Caamal-Velazquez et al. 2007). Total RNA isolated

from the full fruit was used to synthesize the first-strand

cDNA with the GeneRacerTM kit (Invitrogen, CA, USA).

Total RNA from banana fruits was first sequentially treated

with calf intestinal phosphatase and tobacco acid pyro-

phosphatase; a GeneRacerTM RNA oligo was ligated with T4

RNA ligase. First strand cDNA was generated by reverse

transcription using SuperScriptTM II RNase H-Reverse

transcriptase and the GeneRacerTM Oligo dT Primer.

MaCat2 gene fragment was amplified by the polymerase

chain reaction (PCR) using a specific GeneRacerTM 30 nested

primer and a gene-specific MaCat2 forward primer (50-AA

CCGCAACATCGACA-30). MaCat2 complete sequence

was obtained using a specific GeneRacerTM 50 nested primer

and a gene-specific MaCat2 reverse primer (50-TCAGA

TGTTTGCTCTCATGTTGA-30). PCR products were sep-

arated in 1% agarose gels, and isolated using the GENE

CLEAN� II KIT (Q-Biogene). The isolated fragment was

cloned into the pGEM�-T Easy vector (Promega) and

transformed in E. coli strain DH10B by the calcium protocol

transformation (Sambrook and Russell 2001). MaCat2 gene

was sequenced and analyzed using the BLAST database

(http://www.ncbi.nlm.nih.gov/blast/) and the software Clu-

stalW (http://www.ebi.ac.uk/clustalw) (Altschul et al. 1990;

Gish and States 1993). Edited sequences were loaded into the

National Center for Biotechnology Information BLAST

search tool on a network server (www.ncbi.nlm.nih.gov).

Sequence data and phylogenetic analysis

Amino acid sequences of 14 different catalase sequences

from monocots plants were obtained from GENBANK

using BLAST (http://www.ncbi.nlm.nih.gov/blast). An

initial alignment of amino acid sequences was made

using a multiple sequence alignment with the CLU-

STALW software (http://www.ebi.ac.uk/clustalw) and

refined with the software DNASTAR ver. 5.08/5.03

(http://www.ebi.ac.uk/clustalw) and BOXSHADE (http://

searchlauncher.bcm.tmc.edu/multialign/multi-align.html).

The phylogenetic analysis was done using MEGA4

(Tamura et al. 2007). Phylogenetic tree was obtained by the

neighbor-joining (NJ) method (Saitou and Nei 1987) using

a bootstrapped, 1,000-replicate. The evolutionary distances

were computed using the Poisson correction method

(Zuckerkandl and Pauling 1965). Sequence logos of

alignments were generated with WebLogo (Crooks et al.

2004).

RNA gel blot

Total RNA (10 lg) was isolated from vegetative or fruit

tissues of banana plants. Tissues were exposed to different

abiotic stresses and separated. The extracted RNA was

visualized by electrophoresis in 1.5% agarose gel containing

formaldehyde. The nucleic acids were blotted onto a nylon

membrane and hybridized with partial nucleic acid sequence

of MaCat2 as a probe (from 103 to 822 nt) at 54�C. RNA blot

hybridization was performed as described by Sambrook

and Russell (2001). Membranes were washed at 54�C,

according to the protocols of Gene Images AlkaPhos Direct

Labeling and Detection System (Amersham/Biosciences).

The specific band for MaCat2 was visualized by autoradi-

ography at 24�C for 1 h (Hyperfilm ECL, Amersham/

Bioscience).

RT-PCR Analysis of MaCat2 gene

Differential expression of MaCat2 gene was performed by

semiquantitative RT-PCR using specific MaCat2 gene prim-

ers (forward: 50-AACCGCAACATCGACAAC-3, reverse:

50-GCCTATCAGGTGCCCAGGAACGG-30). PCR (Eppen-

dorf Mastercycler) was used to reverse transcribe and amplify

total RNA. Different PCR cycle parameters were tested for

each gene to ensure that the assay was within the linear range

of amplification. The housekeeping 18S gene was amplified

from each sample to normalize the level of each test gene

using 18S universal primers (forward: 50-CGGCTACCAC

ATCCAAGGAA-30, reverse: 5-GCTGGAATTACCGCG

GCT-30). PCR products were run on a 2% agarose gel. Total

RNA quantification was carried out by digital analysis of band

intensity in the gel using the Quantity One software (version

4.6.2.) included in the BioRAD Gel Documentation System

(BioRAD).

Plant Cell Tiss Organ Cult (2012) 109:429–438 431

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Results

Analysis of catalase gene

MaCat2 from banana fruit was obtained by rapid amplifi-

cation of cDNA ends (GeneRacerTM

Kit). BLAST analysis

revealed a protein homologous to plant catalases. The

comparison for the amino acid sequences from different

monocotyledonous catalase plants found in GenBank

database was carried out using the Neighbor-Joining

method (Saitou and Nei 1987). To find the evolutionary

relationship of MaCat2 relative to other plant catalases, a

phylogenetic tree was generated using full-length amino

acid sequences. We obtained 7 catalase protein sequences

related with MaCat2 in clade I (Fig. 1). A catalase A from

Homo sapiens (HsCATA: accession number CAA27721)

was used as an outside group for the rooted tree since the

amino acid sequence of the catalase from Homo sapiens

was significantly different to that of monocotyledonous

plant catalases. The consensus phylogenetic tree obtained

in this study revealed that MaCat2 is related to catalase

type 2 proteins from Elaeis guineensis (EgCAT2, accession

number ACF06566; common name, African oil palm) and

Zantedeschia aethiopica (ZaCAT2, accession number

AAG61140; common name, lily of the Nile). The clade

between MaCat2 and EgCat2 suggests that MaCat2 and

EgCat2 have identical amino acid motif regions. The

amino acid sequence between both catalases indicated the

presence of a conserved hydroperoxidase domain and a

heme binding site. The full length of MaCat2 is 1,479 bp

and contains an open reading frame encoding 493 amino

acid residues (Fig. 2). The mRNA sequence was deposited

in the GenBank database under accession no. EU139298.1.

The sequence indicated the presence of the catalase activity

motif (CAM) with a conserved histidine at position 65

(FARERIPERVVHARGAS), a catalase heme binding site

(HBS) containing a conserved tyrosine at position 350

(RVFAYGDTQ) and a peroxisomal targeting signal

(PTS1) motif (ELRSIWISFLSKCDTSLGQKVANRLNM

RANI) that contains a conserved NRL amino acid residue

at positions 87–89 (Fig. 2). It is known that the amino acid

sequence that is relevant for the import of plant catalases

into peroxisomes seems to reside on the C-terminal end

(PTS1-like motifs) (Kamigaki et al. 2003; Mullen et al.

1997). Comparison of MaCat2 C-terminal regions with

other plant catalases is depicted in Fig. 2. MaCat2 exhibits

the Asn-Arg-Leu tripeptide sequence (at positions

487–489) at the same position than that in Catalase 2 from

Elaeis guineensis and Zantedeschia aethiopica, which

agrees with the suggestion that the internal Ser-Arg-Leu

motif is not the PTS1 of plant catalases (Kamigaki et al.

2003). The amino acids sequence of several catalases from

several plants grouped in clades I and II showed little

difference in amino acid composition of the CAM domain

(Fig. 2). In the cases of the HBS domain and the PTS1

motifs, we observed strong changes in amino acid com-

position (Fig. 2). We identified the CAM, HBS and PTS1

motifs (Fig. 2) that are specific for the catalase protein of

monocots. We identified numerous basic amino acids

(arginine and glutamate) and several uncharged amino

acids (phenyl-alanine, isoleucine, proline, valine and ala-

nine) that appeared to be unique to CAM catalase proteins

with a conserved histidine (Fig. 3a). The HBS motif con-

tains several predominant uncharged amino acids (threo-

nine and glutamine), a basic and acid amino acid (arginine

and aspartate, respectively) and a neutral amino acid

(phenyl-alanine). Also, the HBS motif includes a conserved

tyrosine (Fig. 3b). The C-terminal PTS1 motif, has several

conserved uncharged amino acids (isoleucine, tryptophan,

leucine, alanine and methionine), two acidic amino acids

(glutamate and aspartate), two basic amino acids (lysine

and arginine) and a neutral amino acid (glycine), which are

representative of catalases proteins (Fig. 3c). The PTS1

motif contains conserved (N ? S ? T) RL amino acid

residues (Fig. 3c).

Catalase cold stress gene expression

Effect of abiotic stress on MaCat2 expression was com-

pared between fruits and vegetative tissues of banana

plants. Northern blotting assay and semiquantitative RT-

PCR were used to investigate MaCat2 expression in banana

fruits. Banana fruits were exposed to 10 or 25�C. After 8 h

of exposure to LT stress, different injury symptoms began

to appear in the subepidermal tissues of fruits, which

developed dark-brown streaks (Fig. 4a). Semiquantitative

RT-PCR experiments showed that, at 10�C, MaCat2 tran-

script accumulation increases dramatically in banana fruit

peel, but not so in the fruit pulp (Fig. 4b). Nucleic acid

hybridization corroborated this pattern of MaCat2 gene

expression at 10�C (data not showed). A strong signal of

the MaCat2 gene expression in banana peel was detected

after exposure to 10�C, while a lower signal was detected

when fruits were treated at 25�C. However, in the banana

fruit pulp, the MaCat2 transcript accumulation was dras-

tically lower at 25�C and almost undetectable at 10�C

(Fig. 4b). We observed that the MaCat2 transcript

increased in response to mechanical damage but not to high

temperature exposure (45�C) or during fruit maturation

(Fig. 4c). Likewise, results of the RNA gel blot suggest

that at 10�C MaCat2 transcript accumulation increases in

leaves and decreass in roots of banana plants. Different

results were obtained when banana plants were kept at an

optimal temperature for their development; at 25�C,

MaCat2 transcript accumulation was observed to increase

in roots and decrease in leaves. However, in corm and

432 Plant Cell Tiss Organ Cult (2012) 109:429–438

123

pseudostem tissue of banana plants the expression of

MaCat2 transcript showed little differences at 25 and 10�C

(Fig. 5).

The banana is a typical climacteric fruit and undergoes a

postharvest ripening process characterized by a green

phase followed by a burst in ethylene production that sig-

nals the beginning of the climacteric period. Jin et al.

(2009) conducted a cDNA microarray analysis on banana

fruit 10 days after harvest, which was the time of ripening

initiation by ethylene biosynthesis. They found a total of 16

up-regulated and six down-regulated cDNAs. The cDNAs

identified are involved in signal transduction, amino acid

metabolism, lipid metabolism, proteolysis, citrate biosyn-

thesis and metabolism, and the uptake and transport of

potassium. No catalase genes were found in this report

involving the postharvest ripening process. In the current

paper we present the full-length sequence of a Musa ac-

uminata cv. ‘‘Grand Nain’’ catalase cDNA obtained from

banana fruits, being the first banana fruit catalase identified

up to date.

Plants respond to diverse environmental signals in order

to survive to stress (Pastori and Foyer 2002), because of

which strategies to minimize oxidative damage are uni-

versal features of plant defense responses. Catalases, or

more correctly, hydroperoxidases, are one of the most

studied classes of enzymes involved in the defense against

superoxide radicals (Chelikani et al. 2004).

Many reports suggest that plant genomes have evolved

several catalase genes to satisfy the specific demands upon

various cellular processes (Esaka et al. 1997; Frugoli et al.

Fig. 1 Phylogenetic relationship among amino acid sequences of 16

catalases from different monocots plants. The phylogenetic tree was

inferred using the Neighbor-Joining method. The evolutionary

distances were computed using the Poisson correction method.

Bootstrap percentages are shown on the nodes. Branch lengths are

proportional to the number of amino acid substitutions. A catalase A

from Homo sapiens (HsCATA: accession number CAA27721) was

used as an outside group for the rooted tree. Catalase proteins of clade

I are marked with solid black circles and solid black triangles;

catalase proteins of clade II are marked with solid black squares.

GenBank accession numbers of the sequences are as follows: CATAof Oryza sativa (11375424); CAT2 of Hordeum vulgare (P55308);

CAT2 of Musa acuminata (ABV55108); CAT2 of Elaeis guineensis(ACF06566); CAT2 of Zantedeschia aethiopica (AAG61140); CAT3of Zea mays (P18123); CAT3 of Zea mays (NP_001105416); CAT1 of

Zea mays (CAA42720); CAT1 of Zea mays (P18122); CAT1 of

Hordeum vulgare (P55307); CAT2 of Triticum aestivum (P55313);

CAT1 of Zantedeschia aethiopica (AAF19965); CAT2 of Zea mays(P12365); CAT1 of Festuca arundinacea (CAG23920); CAT1 of

Triticum aestivum (Q43206)

Plant Cell Tiss Organ Cult (2012) 109:429–438 433

123

434 Plant Cell Tiss Organ Cult (2012) 109:429–438

123

1996; Scandalios et al. 1997; Willekens et al. 1994a). The

wide diversity of plant Cat sequences has led to some

discrepancies about the isozyme correlation in phyloge-

netic trees (Luna et al. 2005). In the case of banana, Ma-

Cat2 and EgCat2 have similar sequences and belong to the

same main clade I of the phylogenic tree. ZaCat2 gene

appears to play an exclusive role in scavenging photore-

spiratory H2O2 during senescence and regreening, and it is

phylogenetically related to unspecific Zea mays Cat3 and

Oryza sativa Cat1 (Lino-Neto et al. 2004). Multiple cata-

lase genes in plant genomes have likely arisen by an initial

duplication of a clade 1–type catalase gene, as suggested

by Chelikani et al. (2004). The expression products of these

genes can assemble various tetrameric isozymes, thus, the

regulation of this recombination may allow for the syn-

thesis of tissue-specific catalases (Frugoli et al. 1998; Klotz

and Loewen 2003).

The nucleic acid sequence of the banana catalase

showed a similitude with the monocot Elaeis guineensis

Cat2 (EgCat2). This MaCat2 gene contained a hydroper-

oxidase catalytic domain and a heme binding site similar to

those of other plant Cat2 genes (Chelikani et al. 2004;

Klotz et al. 1997). To date, experimental evidence obtained

from eukaryotic catalases has indicated that sorting to the

peroxisomal matrix is made possible by a peroxisomal

targeting signal (PTS) in the form of a C-terminal poly-

peptide (Mullen et al. 1997). CAT2 from cotton seed has a

PTS1 (-SRLNVRPSI) that is important for its subcellular

localization in the peroxisomal matrix. In contrast, MaCat2

exhibits the Asn-Arg-Leu tripeptide in the same position to

that reported in ZaCat2, which agrees with the suggestion

that plant catalase PTS1 is not the internal Ser-Arg-Leu

motif but the C259 terminal degenerated tripeptide Pro-

Ser/Thr/Asn-Met/Ile (Mullen et al. 1997). Differences

found in the present study between the PTS of MaCat2 and

that of other catalases suggest that neither the PTS1

reported by Gonzalez (1991) nor the PTS1 tripeptide pro-

posed by Mullen et al. (1997) are exclusively necessary for

targeting catalase to peroxisomes. Indeed, Kamigaki et al.

(2003) reported that the C-terminal 10-amino acid

sequence, comprising both previously suggested PTS1, was

not required for the import into peroxisomes. According to

Fig. 3 Sequence logos of several plant catalases generated by the

WebLogo program (http://weblogo.threeplusone.com) showing con-

tributions of individual amino acid positions to the overall CAM,

HBS and PTS1 motifs. In the logo, the letters are sorted so that the

most common one is on top, the height of each letter is made

proportional to its frequency, and the height of the entire stack is

adjusted to signify the ‘information content’ (measured in bits) of the

sequences at that position. The logo displays both significant residues

and subtle sequence patterns. a CAM catalase activity motif, b HBS

catalase heme binding site, c PTS1 peroxisomal targeting signal 1

Fig. 2 Alignment of the complete deduced amino acid sequence of a

catalase 2 from banana fruits (MaCat2) with other 7 catalases with

inducible transcription gene expression reported (see reference in the

NCBI GenBank database by searching the accession numbers of each

amino acid sequence). Deduced amino acid sequence indicates the

presence of a catalase activity motif (CAM), with conserved

histamine 65, and a catalase heme binding site (HBS), which contains

the conserved tyrosine 350. Catalase contains a peroxisomal targeting

signal (PTS1). GenBank accession numbers of the sequences are as

follows: CATA of Oryza sativa (11375424); CAT2 of Hordeumvulgare (P55308); CAT2 of Musa acuminata (ABV55108); CAT2 of

Elaeis guineensis (ACF06566); CAT2 of Zantedeschia aethiopica(AAG61140); CAT3 of Zea mays (P18123); CAT3 of Zea mays(NP_001105416); CAT1 of Zea mays (CAA42720); CAT1 of Zeamays (P18122); CAT1 of Hordeum vulgare (P55307); CAT2 of

Triticum aestivum (P55313); CAT1 of Zantedeschia aethiopica(AAF19965); CAT2 of Zea mays (P12365); CAT1 of Festucaarundinacea (CAG23920); CAT1 of Triticum aestivum (Q43206)

b

Plant Cell Tiss Organ Cult (2012) 109:429–438 435

123

these authors, the effective PTS1 tripeptide is the internal

degenerated motif Gln-Lys-Leu/Ile/Val, located 11–13

amino acid residues from the C-terminal end; this finding

agrees with the results of the present work, this motif being

fully conserved in MaCat2. Our results indicate that Ma-

Cat2 may also be participating in the signal transduction

cascade caused by low temperature and mechanical dam-

age. Banana fruits exposed to LT or mechanical damage

treatments showed increased MaCat2 transcript levels, an

increment that was absent from fruits experiencing high

temperature stress or during the maturation process. There

are several studies in plants that support this result. Prasad

et al. (1994) and Willekens et al. (1994a) found an

increased level of catalase transcript after exposure to low

temperatures in maize seeds and in Nicotiana plumbagin-

ifolia plants, respectively. In A. thaliana, it is interesting to

note that expression of genes encoding the cytosolic or

peroxisomal catalases at low temperature were either

unchanged (CAT1) or down regulated (CAT3), catalase 2

(CAT2) being an exception and is clearly up regulated after

all three treatments, i.e. Cold/Light, Cold/Dark and Dark

conditions (Soitamo et al. 2008). In Oryza sativa leaves

after cold exposures in the light, periods of catalase defi-

ciency were short in comparison with the same in

cucumber (Cucumis sativus). In addition, alternative anti-

oxidants increased, H202 did not accumulate and major

photo oxidative injury was not observed in rye (Volk and

Feierabend 1989). Currently, cucumber is the only plant for

which an increase of the H202 content has been described

after experimental catalase depletion (Omran 1980). Also

Sakajo et al. (1987) reported an increment of the catalase

mRNA expression caused by wounding sweet potato

tuberous root tissue. In Zea mays embryos and leaves, all

Cat genes expression were up regulated in response to

wounding and jasmonic acid (JA), raising the possibility

that JA and wounding may share a common signal trans-

duction pathway in up regulating Cat mRNA in immature

embryos (Guan and Scandalios 2000). In addition, Guan

and Scandalios (2000) found that Cat1 and Cat3 transcript

Fig. 4 Differential accumulation of MaCat2 transcripts in banana

fruits in response to low temperature. a Chilling injury symptoms

after 8 h to low temperature treatment in banana fruits using 10 or

25�C (room temperature). b Semiquantitative RT-PCR using specific

primers for the MaCat2 gene. Banana fruits were treated at 10 or

25�C. mRNA was extracted for each stress treatment, and RT-PCR

reaction was carried out. Specific primers for the 18S gene were used

as control; c Different stresses produce a differential accumulation of

the MaCat2 transcripts in banana fruits. Banana fruits were submitted

at different stresses and total RNA was isolated from each treatment,

blotted onto a nylon membrane and hybridized with the MaCat2probe. rRNA was used as a loading control. C Banana fruits without

treatment; M Banana fruit during regular maturation process; MDBanana fruit with mechanic damage treatment; and HT Banana fruit

with high temperature treatment

Fig. 5 RNA gel blot analysis using different banana plant tissues.

Banana plants were treated at 10 or 25�C. Total RNA was isolated

from each treated tissue, blotted onto a nylon membrane and

hybridized with partial nucleic acid sequence of MaCat2. rRNA

was used as a loading control. L banana leaves; PS banana

pseudostem; P banana corm and R banana roots

436 Plant Cell Tiss Organ Cult (2012) 109:429–438

123

accumulation increased in response to wounding in both

wild-type and mutant leaves deficient in abscisic acid

(ABA), implying that Cat1 and Cat3 induction in response

to wounding is not mediated by ABA in leaves. Further

experiments are needed to understand why chilling-tolerant

plants in the temperature range of 10–15�C are less sen-

sitive than susceptible species.

The study of the mechanisms that regulate the expres-

sion of these peroxisomal metabolism enzymes will pro-

vide further understanding of the physiological significance

of peroxisomes as a source of signaling molecules in

plants. This information could be valuable to design new

molecular strategies directed to improve the tolerance of

plants to different abiotic stresses.

Acknowledgments The authors acknowledge support for the pres-

ent work provided by CONACYT (SAGARPA-C01-2002-1714;

Ciencia Basica SEP-CONACYT 59097) and the International Foun-

dation for Science (C/3959-1). We are thankful to Fernando Contreras

for his agronomic technical support, and to Bartolome Chı for tech-

nical support.

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