Journal of Environmental Biology �January, 2009�

© Triveni Enterprises, Lucknow (India) J. Environ. Biol.

ISSN : 0254-8704 30(1), 93-98 (2009)

http : //www.jeb.co.in info@jeb.co.in

Cloning and expression analysis of an aldehyde oxidase gene in

Arachis hygogaea L.

Lixia Yang, Jianhua Liang, Haihang Li and Ling Li*

Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Science,

South China Normal University, Guangzhou - 510631, P.R. China

(Received: October 16, 2007; Revised received: February 10, 2008; Accepted: March 05, 2008)

Abstract: Aldehyde oxidase (AO) plays important role in plant hormone biosynthetic pathways, such as abscisic acid (ABA) and indole-

3-acetic acid (IAA). The enzyme catalyzes the last step of the pathways. In this study, a full-length cDNA encoding an aldyhyde oxidase

was cloned and sequenced from leaves of peanut by RT-PCR, RACE-PCR and genomic DNA walking methods. The full-length cDNA,

designated as Arachis hygogaea L. aldehyde oxidase1 (AhAO1), consists of an open reading frame of 4131bp, a 326 bp 5′ untranslated

region and a 128 bp 3′ untranslated region including a poly (A) tail of 21 nucleotides. The gene encodes a polypeptide of 1377 amino acids

with a calculated molecular weight of 150 kDa and an isoelectric point (pI) of 6.99. Analysis of amino acid sequence of AhAO1 shows that

it had 61%, 59% and 55% identity with the AOs from tomato, Arabidopsis and maize, respectively. The peanut AO polypeptide contains

consensus sequences for iron-sulfur centers and a molybdenum cofactor (MoCo)-binding domain. Semi-quantitative RT-PCR analysis

showed that AhAO1 expression was higher in leaves than in roots of peanut.

Key words: Cloning, Expression, Aldehyde oxidase, Arachis hypogaea L.

PDF of full length paper is available with author (*lilab@scnu.edu.cn)

Introduction

Aldehyde oxidase (AO) catalyzes the oxidation of a variety

of aldehydes and N-containing heterocyclic compounds in the

presence of O2 or certain redox dyes (Hall and Krenitsky, 1986).

AOs belong to the family of molybdenum-containing protein like

xanthine dehydrogenase (XD) and sulfite oxidase (Hille and Massey,

1985). These enzymes possess a short oxidoreductive chain

characterized by four oxidation centers, two iron-sulfur clusters, a

flavine cofactor, and a molybdopterin cofactor (MoCo) (Barber et al.,

1982). AO has been characterized extensively in animals and

microorganisms, where it has been implicated in the detoxification of

environmental pollutants and xenobiotics (Bauer and Howard, 1991).

In higher plants, much of the interest in AO stems from its involvement

in the biosynthesis of abscisic acid (Leydecker et al., 1995) and

indole acetic acid ( Koshiba and Matsuyama, 1993).

ABA is a key regulator in seed development, root growth,

stomatal aperture in higher plants (Zeevaart and Creelman, 1988).

It is also involved in adaptation of plants to various stresses (Zeevaart

and Creelman, 1988), possible the same as other plant growth

regulators (Kim et al., 2006; Chandra et al., 2007; Tilki, 2008). The

regulation of these processes by ABA is partially due to de novo

synthesis of ABA. Endogenous ABA increases upon stress conditions,

most notably under drought and salinity (Munns and Cramer, 1996).

Application of exogenous ABA has similar effects to plants as stress

treatments and increases plant tolerance to environmental stresses

(Fedina et al., 1994; Welbaum et al., 1997). Elucidation of ABA

biosynthetic pathway, related genes and enzymes are important for

the control and regulation of ABA-mediating plant responses.

Two enzymes, involved in the early steps of ABA synthesis in

chloroplasts, have attracted much attention. Zeaxanthin epoxidase

(ZEP) converts zeaxanthin to violaxanthin by a two-step epoxidation

reaction (Marin et al., 1996), and 9-cis-epoxycarotenoid dioxygenase

(NCED) catalyses the oxidative cleavage of 9-cis-xanthophylls to

xanthoxin (Cutler and Krochko, 1999). Over-expression of ZEP in

transgenic tobacco resulted in enhanced seed dormancy (Frey et

al., 1999). Over-expression of the AtNCED3 in Arabidopsis resulted

in increased ABA levels (Iuchi et al., 2001). It was suggested that

NCED catalyses the critical step in the regulation of ABA biosynthesis

(Qin and Zeevaart, 1999; Iuchi et al., 2000).

AO catalyses the final step of ABA biosynthesis. Plant AO

has only been purified from coleoptiles of maize (Koshiba et al.,

1996). Based on the amino acid sequence of the purified AO, two

cDNAs were cloned from maize (Sekimoto et al., 1997). Four cDNAs

(Arabidopsis aldehyde oxidase, AAO1, 2, 3 and 4) from Arabidopsis

(Sekimoto et al., 1998), three cDNAs from pea (Zdunek-Zastocka,

2008) three putative AO genes and two AO pseudogenes from

tomato (Ori et al., 1997, Min et al., 2000) have been reported. Plant

AO is a protein family with similar amino acid sequence, but different

in electrophoretic mobility, substrate specificity, and subunit

composition. An AO polymorphism has been observed in potato

bubers (Rathe, 1974), maize coleoptiles (Koshiba et al., 1996),

Arabidopsis (Akaba et al., 1999), ryegrass (Sagi et al., 1998),

barley (Omarov et al., 1999) and in pea (Zdunek and Lips, 2001).

Peanut (Arachis hypogaea L.) is an important crop. Drought

is one of the major abiotic stresses that limit its growth and production.

The role of AO in the ABA biosynthesis pathways was only well

Journal of Environmental Biology �January, 2009�

Yang et al.

investigated in Arabidopsis. In the present paper, we describe the

molecular cloning of a full-length Arachis hygogaea L. aldehyde

oxidase1 (AhAO1) in attempt to clone an AO gene involved in

ABA biosynthesis, by reverse transcription-polymerase chain

reaction (RT-PCR), rapid amplification of cDNA ends (RACE)

PCR and genomic walking methods. Its expression in peanut

leaves and roots is tested by semi-quantitative RT-PCR to

understand the function and role for peanut during growth and

development.

Materials and Methods

Plant material: Seeds of peanut (Arachis hypogaea L. cv. YueYou

7) were planted in culture medium (vermiculite: perlite: soil, 1: 1: 1)

and grown in a growth chamber with 16 hr of light (200 µmolm-2s-1)

at 26oC and 8 hr of darkness at 22oC. Plants were irrigated daily

with half-strength Murashige and Skoog nutrient solution (Murashige

and Skoog, 1962).

RNA extraction: Total RNA was extracted from peanut leaves (10

days after planting) using a modified phenol–chloroform method

(Vanlerberghe and McIntosh, 1994). 80 mg tissue was ground in

liquid nitrogen to a fine powder using a mortar and pestle. The

ground tissue was transferred to a tube containing 800 µl extraction

buffer (4 mol l-1 guanidine isothiocyanate, 25 mmol l-1 sodium citrate,

200 mmol l-1 sodium acetate(pH4.0), 0.5% (w/v) lauroyl sarcosine,

and 1%(v/v) b-mercaptoethanol). The homogenate was extracted

with phenol/chloroform (1: 1, v/v) on ice for 30 min. After centrifugation

at 12 000 g at 4oC for 20 min, the aqueous phase was transferred

to a fresh RNase-free tube, mixed with equal volume of isopropyl

alcohol and kept on ice for 1 hr. RNA was pelleted and washed

twice with 75% (w/w) cold ethanol. The pellet was resuspended in

diethypyrocarbonate (DEPC) water.

Each RNA sample was quantified with a spectrophotometeric

method (Sambrook et al., 1989). The A260/A280 values of the

RNA samples were all greater than 1.8.

RT-PCR amplification of the conserved region: cDNA was

synthesized from total RNA (2 µg) using SuperScriptTM III RNase

H- Reverse Transcriptase (Invitrogen) and an oligo dT18

primer.

For amplification of a specific fragment of AO gene from peanut, two

primers (AOF3: 5′ -GGI GGI GG/CI TTT/C GGI GGI AA-3′ and

AOR3: 5′ -CAT T/CTG T/CTT IAC T/CTT IGT CCA-3′ ) were

synthesized based on the conserved regions of reported AO genes

(Sekimoto et al., 1998). The fragment between the two conserved

domains was amplified by PCR using the two primers (PCR

conditions: 94oC for 1 min, 52oC for 1 min and 72oC for 2 min, 40

cycles). PCR amplified fragment was cloned into pMD 18-T vector

and confirmed by sequencing.

RACE-PCR amplification for 3′′′′ -end: The 3′ -end of the gene was

obtained using the GeneRacer kit (Invitrogen) according to the

manufacture’s instructions with a gene-specific primer (GSP) of 3AO-

outer (5′ -GGA TTA TCT CGT CTA CCA GTT GTG-3′ ) and a nested

primer of 3AO-inner (5′ -CTC GGA TGG ATC GGT TGT TGT TG-3′ ).

PCR amplification 5′′′′ -end: The first 5′ extended cDNA (RACE-

PCR1) was obtained by amplification with a forward degenerate

primer (AOF, 5′ -CAA/G TGC/T GGA/G/C/T TTC/T TGC/T ACA/

G/C/T CCI GGA/G/C/T ATG-3′ ) and a specific primer (yang 1, 5′-CAC TGT AAG TAA TCT TCA TGG G-3′ ). AOF corresponded to

the conserved peptide QCGFCTPGM in all known plant AOs was

designed. The second 5′ extended cDNA (RACE-PCR2) was

obtained with the forward degenerate primer (AOF4, 5′ -GAG/A

GGA/G/C/T GG A/G/C/T TGC/T GGA/G/C/T GCI TGC/T-3′ ) and

a specific primer (yang 2, 5′ -TTT GGA GAA TCC GGA AGG CGG

ATC C-3′).

Genomic walking for the 5′′′′ -end: The 5′ end of the sequence

was obtained from genomic DNA using Genome Walker kit

(Clontech). The genomic DNA was isolated according to DNA

Extraction kit (Tiangen, China). The DNA was digested with restriction

enzyme (Dra I) and ligated to short adaptor sequences. The first

PCR with primers AP1 (5′ -GTA ATA CGA CTC ACT ATA GGG C-3′)and AOP1 (5′ -CAC CTT CAA GTT CAT TGA AAA CCC CAT

CAG-3′ ) was performed as follows: 94oC, 2 min, 7 cycles of 30 s at

94oC, 3 min at 62oC, followed by 33 cycles of 30 s at 94oC, 3 min at

58oC, and another period of 58oC for 10 min after the final cycle.

The 1 µl genome-walking library was used as template in the first

PCR. The nested PCR with primers AP2 (5′ -ACT ATA GGG CAC

GCG TGG T-3′ ) and AOP2 (5′ -ACC AGA ACA AGG TCC TTT

ATC CTT ATG CCT-3′ ) was performed in the same conditions as

in the first PCR and 1µl product of the first PCR was used as

Table - 1: Identity (% ) among aldehyde oxidases and xanthine dehydroginase

AhAO1 AAO1 AAO2 AAO3 AAO4 TAO1 TAO2 TAO3 ZmAO1 ZmAO2

AAO1 58.7

AAO2 60.9 65.5

AAO3 60.1 60.4 61.0

AAO4 59.4 60.4 60.4 73.3

TAO1 59.7 57.2 58.4 58.5 58.1

TAO2 60.2 58.2 60.1 59.2 59.5 82.3

TAO3 60.9 58.8 60.8 59.5 59.8 83.2 85.0

ZmAO1 54.6 55.2 55.5 55.2 54.0 54.9 57.6 57.3

ZmAO2 54.5 55.4 56.0 54.8 54.2 55.7 56.6 56.9 83.8

AXD1 29.2 28.3 28.7 28.4 28.0 28.9 28.5 29.0 28.6 29.6

94

Journal of Environmental Biology �January, 2009�

Fig. 1: Structural organization of AhAO1 cDNA. The shaded and white boxes indicated the protein coding regions and the 5′ - or 3′ - untranslated regions,

respectively. The thin lines shown in the lower part of figure represent reverse transcription-polymerase chain reaction, RACE-polymerase chain reaction

or genome walking products

Cloning and expression of an aldehyde oxidase gene

template. For amplification of the 5′ cDNA sequence of AhAO1, PCR

was performed with the primers yang 3 (5′ -ATG GAA GTG AAG

AAC AAC ATC-3′) and yang 4 (5′ -TCT TCC GCC TCT GCT

GCT GTC-3′).

Cloning, DNA sequencing, and sequence analysis: All the

amplification products were cloned into a TA cloning vector PMD

18-T (TaKaRa), and sequenced in a 3730 DNA analyzer by

Invitrogen (Guangzhou, China). Multiple alignment of the amino

acid sequences was carried out using the Clustal W program in

BioEdit software. For sequence identity and phylogenetic analysis,

we used megalign software (DNASTAR).

Semi-quantitative RT-PCR analysis: To estimate AhAO1

expression in peanut organs, semi-quantitative RT-PCR analysis

was performed. Extraction of total RNA is the same as described

above. To avoid amplification of peanut AhAO1 genomic DNA in

semi-quantitative RT-PCR analysis, the isolated RNA was treated

with RNase-free DNase I (TaKaRa) at 37oC for 15 min. The reaction

mixture was extracted with phenol: chloroform: isoamyl alcohol (25:

24: 1, v/v) followed by ethanol precipitation. The RNA samples

were also visualized by ethidium bromide (EtBr) staining after

agarose gel electrophoresis. The first-strand cDNA was synthesized

from 20 ng of total RNA using reverse transcriptase (superscriptTM

III, Invitrogen). For gene-specific primers, yang 5 (5′ -AGT TGC

AGA AGG AAA TGG GAT C-3′) and yang 6 (5′ -ATC CGT TCC ATC

TTT GTT GCT C-3′) were used to amplify a 548 bp fragment. As an

internal loading control, primers 18S-F (5′ -ATT CCT AGT AAG

CGC GAG TCA TCA G-3′) and 18S-R (5′ -CAA TGA TCC TTC

CGC AGG TTC AC-3′) specific to a peanut 18S rRNA gene (Bhagwat

et al., 2001) were used to amplify a fragment of 226 bp. The PCR

conditions for AhAO1 was 94oC for 5 min followed by 28 cycles of

94oC for 30 s, 52oC for 45 s, and 72oC for 1 min. The PCR reaction

for 18S was performed at 94oC for 5 min followed by 25 cycles of

94oC for 30 s, 60oC for 45 s, and 72oC for 1 min. PCR products were

visualized by EtBr staining after agrarose gel electrophoresis.

Results and Discussion

Cloning and characterization of AhAO1: Fig.1 illustrated the

PCR amplification strategy for the cloning of the peanut AO gene.

Amino acid sequence analysis of AOs of Arabidopsis, maize and

tomato reveals that two domains, GGGFGGK and WTKVKQM, are

highly conserved in these genes. They were used to design primers,

AOF3 and AOR3, for PCR amplification of the peanut AO gene. A

759 bp fragment was amplified from peanut leaf cDNA and was

confirms to be part of the AO gene by DNA sequencing. The 3′ end

was obtained by RACE-PCR. To obtain the 5′ end, two PCR

reactions were tried, with two degenerate primers and two gene

specific primers. However, we failed to obtain the full-length sequence

from cDNA using the Gene Racer kit and SMART RACE cDNA

Amplification kit (Clontech). A genomic walking method was employed

to clone the 5′ end sequence of AhAO1 from genomic DNA. By

using two gene specific primers (yang 3 and yang 4), we

successfully cloned the 5′ end sequence and got a full-length

sequence of the AhAO1. All primers used, except for yang AOP1,

AOP2, AP1 and AP2, are indicated in Fig. 2. The full sequence in

amino acid of the gene is shown in Fig. 2.

The full-length of AhAO1 cDNA is 4585 bp and contains a

4131 bp open reading frame (ORF), a 5′ untranslated region (5′UTR) of 326 bp and a 3′ UTR of 128 bp. AhAO1 encodes a

polypeptide of 1377 amino acids with a calculated molecular weight

of 150 kDa and an isoelectric point (pI) of 6.99.

Analysis of the deduced amino acid sequences of AhAO1:

Sequence analysis shows that AhAO1 has 61%, 59% and 55%

identity in amino acid sequence with the AOs from tomato, Arabidopsis

and maize, respectively (Table 1). Alignment of amino acid sequences

of AhAO1 with 9 AOs registered in Genebank are shown in Fig. 2

and their phylogenetic relationship was shown in Fig. 3. Predicted

protein for AhAO1 has a high level of similarity to plant AOs and can

be aligned along their entire protein sequences (Fig. 2). As in

other plant and animal AOs (Koshiba et al., 1996), three special

domains for binding cofactor or prosthetic groups could be identified

in the peanut AhAO1, two N-terminal iron-sulfur centers and a

molybdopterin binding domain. One of the two iron-sulfur centers

with consensus sequences (CX4CX

2CXnC) is located between

aa 54-85. The second putative iron-sulfur center is between aa

123-178. Each of the two centers has four cysteines, which may

contribute to the structure and functions of the iron-sulfur center.

95

Journal of Environmental Biology �January, 2009�

Yang et al.

Fig

. 2: A

lignm

ent o

f ded

uced

am

ino

acid

seq

uenc

e of

the

pean

ut a

ldeh

yde

oxid

ases

with

AO

s fr

om A

rabi

dops

is, t

omat

o an

d m

aize

and

put

ativ

e ac

tive

mo

tifs.

The

seq

uenc

es

wer

e al

igne

d us

ing

the

Bio

Edi

t

prog

ram

. The

cD

NA

seq

uenc

es u

sed

for a

min

o ac

id tr

ansl

atio

n an

d G

enB

ank

acce

ssio

n nu

mbe

rs a

re: A

hAO

1 (E

U18

3360

) fro

m p

eanu

t; A

AO

1 (A

B00

5804

), A

AO

2 (A

B00

5805

), A

AO

3 (A

B01

6602

) and

AA

O4

(AB

0372

71) f

rom

Ara

bid

opsi

s; T

AO

1 (A

F25

880

8), T

AO

2 (A

F25

8809

) and

TA

O3

(AF

258

810)

fro

m to

mat

o; Z

mA

O1

(D88

451

) an

d Z

mA

O2

(D88

452)

from

mai

ze. T

he th

in li

nes

repr

esen

ted

the

prim

ers

and

puta

tive

activ

e m

otifs

. a, b

1, b

2: c

yste

ine

s re

port

ed

to b

e in

volv

ed in

the

form

atio

n o

f iro

n-su

lfur c

ente

rs. c

-g: f

ive

mot

ifs in

volv

ed in

Mo

Co

bin

ding

. h: p

ropo

sed

pept

ide

reg

ions

as

subs

trat

e bi

ndin

g

96

Journal of Environmental Biology �January, 2009�

The consensus motifs of the two iron-sulfur centers were also

found in the corresponding regions of AOs from tomato (Ori et al.,

1997), maize (Sekimoto et al., 1997) and Arabidopsis (Sekimoto

et al., 1998).

Although the presence of flavin adenine dinucleotide (FAD)

had been demonstrated in purified AO from coleoptiles of maize

(Koshiba et al., 1996), the FAD-binding domain described by Correll

et al. (1993) cannot be identified in AhAO1. The same results were

reported in Arabidopsis AOs (Sekimoto et al., 1998).

The C-terminal domain participates for the MoCo and

substrate binding (Sekimoto et al., 1998) were also identified in the

AhAO1. Five motifs for MoCo binding sites, MoCo 1 (aa 839-845),

MoCo 2 (952-961), MoCo 3 (1080-1082), MoCo 4 (1124-1129)

and MoCo 5 (1302-1307), are found in the sequence of AhAO1 (Fig.

2 c, d, e, f and g). A substrate binding site in plant AOs was proposed

by Sekimoto et al. (1998). A domain (IMPARLVSG, aa 919-927) with

similar sequences to Arabidopsis AAO3 (IMPRNIMGP, aa 878-886)

and tomato TAO1 (IIPSYLMNT, aa 906-914) was considered to be

the substrate binding sequence in AhAO1 (Fig. 2, h).

Gene expression analysis of AhAO1 in peanut roots and

leaves: Semi-quantitative RT-PCR analysis showed that AhAO1

was expressed both in roots and leaves (Fig. 4). The expression

level of AhAO1 is slightly higher in leaves than in roots.

Four AOs isoforms (AOα, AOβ, AOγ, AOδ) were identified

in Arabidopsis thaliana and are all in dimmers. AOα, AOβ and AOδare homodimers of AAO1, AAO2 and AAO3 products, respectively,

while AOβ is a heterodimer of AAO1 and AAO2 products (Akaba et

al., 1999; Seo et al., 2000). Studies on Arabidopsis mutant superroot1

(sur1), an auxin-overproducing mutant, have shown that AAO1

has a higher substrate preference for indole-3-aldehyde. AAO1

activity is significantly higher in sur1 mutant seedlings than in wild

type (Seo et al., 1998). AO isoforms can efficiently oxidize abscisic

aldehyde to abscisic acid in Arabidopsis rosette leaves (Seo et al.,

2000), in roots of barley (Omarov et al., 2003) and in pea (Zdunek-

Zastocka et al., 2004). Drought-induced increase of abscisic

aldehyde oxidase activity is observed in leaves of Arabidopsis by

Bittner et al. (2001). When the Arabidopsis rosette leaves are

detached and exposed to dehydration, the expression of AAO3

mRNA increases rapidly, but AOδ activity is unaffected (Seo et al.,

2000). An ABA-deficient Arabidopsis mutant, aao3, was maps at the

AAO3 locus. A wilty phenotype of aao3 mutant could be restored to

wild type when AAO3 gene was introduced into it (Seo et al., 2000).

It was suggested that AAO1 may be responsible for IAA biosynthesis,

while AAO3 for ABA biosynthesis in Arabidopsis. The gene AhAO1

we cloned from peanut has 58.7, 60.9, 60.1 and 59.4% identities,

in amino acid sequence, with Arabidopsis AAO1, AAO2, AAO3 and

AAO4, respectively (Table 1). Although it showed slightly higher

identity with AAO2 and AAO3, compared to AAO1 and AAO4, we

are unable to identify the type and function of AhAO1 at this point.

Local sequence analysis for all possible function motifs could not

distinguish them, too. AhAO1 belongs to a multigene family coding

for AOs which act on variable substrates. Production of transgenic

Arabidopsis with AhAO1 is progressing and will be useful for

understanding the function of the gene of peanut.

Acknowledgments

This research was supported by a grant (03098) of the

key project on scientific and technological research from Ministry of

Education of P. R. China and the national natural sciences foundation

of China (grant nos. 30771297 to L. Li).

References

Akaba, S., M. Seo, N. Dohmae, K. Takio, H. Sekimoto, Y. Kamiya, N.

Furuya, T. Komano and T. Koshiba: Production of homo- and hetero-

dimeric isozymes from two aldehyde oxidase genes of Arabidopsis

thaliana. J. Bio. Chem., 126, 395-401 (1999).

Barber, M.J., M.P. Coughlan, K.V. Rajagopalan and L.M. Siegel: Properties

of the prosthetic groups of rabbit liver aldehyde oxidase: A comparison

of molybdenum hydroxy lase enzymes. Biochemistry , 21, 3561-

3568 (1982).

Bauer, S.L. and P.C. Howard: Kinetics and cofactor requirements for the

nitroreductive metabolism of 1-nitropyrene and 3-nitrofluoranthene

by rabbit liver aldehyde oxidase. Carcinogenesis, 12, 1545-1549

(1991).

Bhagwat, A.S., T.G. Krishna, N. Jawali and R.K. Mitra: Cloning and

characterisation of a ribosomal RNA gene repeat unit from groundnut.

Plant Cell Rep., 20, 193-197 (2001).

Bittner, F., M. Oreb and R.R. Mendel: ABA3 is a molybdenum cofactor

sulfurase required for activation of aldehyde oxidase and xanthine

dehydrogenase in Arabidopsis thaliana. J. Biol. Chem., 276, 40381-

40384 (2001).

Chandra, A., A. Anand and A. Dubey: Effect of salicylic acid on morphological

and biochemical attributes in cowpea. J. Environ. Biol., 28, 193-196

(2007).

Correll, C.C., M.L. Ludwig, C.M. Bruns and P.A. Karplus: Structural

prototypes for an extended family of f lavoprotein reductases:

C o m pa r i s o n o f p h t ha l a t e d i o x y ge n a s e re d u c t a s e w i t h

ferredox in reductase and fer redox in . Protein Sci . , 2 , 2112-

2133 (1993).

Cloning and expression of an aldehyde oxidase gene

Fig. 4: Expression of AhAO1 in different tissues of peanut analyzed by

semi-quantitative RT-PCR

organ root leaf

AhAO1

18S rRNA

organ root leaf

AhAO1

18S rRNA

Fig. 3: Phylogenetic relationship of aldehyde oxidases and xanthine

dehydrogenases from different plant species based on amino acid sequence

analysis. The Gen Bank accession numbers for AXD1 is TI0235 and

others have been indicated in Fig. 2

97

Journal of Environmental Biology �January, 2009�

Cutler, A.J. and J. E. Krochko: Formation and breakdown of ABA. Trends.

Plant Sci., 4, 472-478 (1999).

Fedina, I.S., T.D. Tsonev and E.I. Guleva: ABA as a modulator of the

response of Pisum sativum to salt stress. J. Plant Physiol., 143,

245-249 (1994)

Frey, A., C. Audran, E. Marin, B. Sotta and A. Marion Poll: Engineering

seed dormancy by the modification of zeaxanthin epoxidase gene

expression. Plant Molecular Biology, 39, 1267-1274 (1999).

Hall, W.W. and T.A. Krenitsky: Aldehyde oxidase from rabbit liver: Specificity

toward purines and their analogs. Arch. Biochem. Biophys., 251, 36-

46 (1986).

Hille, R. and V. Massey: Molybdenum-containing hydroxylases: Xanthine

oxidase, aldehyde oxidase and sulphite oxidase. In: Molybdenum

enzymes (Ed.: T.G. Spiro). Wiley Interscience, New York. pp. 443-518

(1985).

Iuchi, S., M. Kobayashi, T. Taji, M. Naramoto, M. Seki, T. Kato, S. Tabata,

Y. Kakubari and K. Yamaguchi-Shinozaki: Regulation of drought

to lerance by gene manipula t ion of 9-cis-epoxycarotenoid

dioxygenase, a key enzyme in abscis ic ac id biosynthesis in

Arabidopsis. The Plant J., 27, 325-333 (2001).

Iuchi, S., M. Kobayashi, K. Yamaguchi-Shinozaki and K. Shinozaki: A

stress-induc ible gene for 9-cis-epoxycarotenoid d ioxygenase

involved in abscisic acid biosynthesis under water stress in drought-

tolerant cowpea. Plant Physiol., 123, 553-562 (2000).

Kim, S.K., T.K. Son, S.Y. Park, I.J. Lee, B.H. Lee, H.Y. Kim and S.C. Lee:

Influences of gibberellin and auxin on endogenous plant hormone

and starch mobilization during rice seed germination under salt stress.

J. Environ. Biol., 27, 181-186 (2006).

Koshiba, T. and H. Matsuyama: An in vitro system of indole-3-acetic acid

formation from tryptophan in maize (Zea mays) coleoptile extracts.

Plant Physiol., 102, 1319-1324 (1993).

Koshiba, T., E. Saito, N. Ono, N. Yamamoto and M. Sato: Purification and

properties of flavin- and molybdenum-containing aldehyde oxidase

from coleoptiles of maize. Plant Physiol., 110, 781-789 (1996).

Leydecker, M., T. Moureaux, Y. Kraepiel, K. Schnorr and M. Caboche:

Molybdenum cofactor mutants, specif ically impaired in xanthine

dehydrogenase act ivit y and abscis ic acid biosynthesis,

simultaneously overexpress nitrate reductase. Plant Physiol., 107,

1427-1431 (1995).

Marin, E., L. Nussaume, A. Quesada, M. Gonneau, B. Sotta, P. Hugueney,

A. Frey and A. Marion-Poll: Molecular identification of zeaxanthin

epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic

acid biosynthesis and corresponding to the ABA locus of Arabidopsis

thaliana. EMBO Journal, 15, 2331-2342 (1996).

Min, X.J., K. Okada, B. Brockmann, T. Koshiba and Y. Kamiya: Molecular

cloning and expression patterns of three putative functional aldehyde

oxidase genes and isolation of two aldehyde oxidase pseudogenes

in tomato. Biochim. Biophys. Acta, 1493, 337-341 (2000).

Munns, R. and G.R. Cramer: Is coordination of leaf and root growth mediated

by abscisic acid? Plant and Soil, 185, 33-49 (1996).

Murashige, T. and F. Skoog: A revised medium for rapid growth and bio-assay

with tobacco tissue cultures. Physiol. Plantarum., 15, 473-497 (1962).

Omarov, R.T., S. Akaba, T. Koshiba and S.H. Lips: Aldehyde oxidase in

roots, leaves and seeds of barley (Hordeum vulgare L.). J. Exp.

Bot., 50, 63-69 (1999).

Omarov, R.T., D. Drager, R. Tischner and H. Lips: Aldehyde oxidase

isoforms and subunit composition in roots of barley as affected by

ammonium and nitrate. Physiol. Plantarum., 117, 337-342 (2003).

Ori, N., Y. Eshed, P. Pinto, I. Paran, D. Zamir and R. Fluhr: TAO1, a

representative of the molybdenum cofactor containing hydroxylases

from tomato. J. Biol. Chem., 272, 1019-1025 (1997).

Qin. X. and J.A.D. Zeevaart: The 9-cis-epoxycarotenoid cleavage reaction

is the key regulatory step of abscisic acid biosynthesis in water-

stressed bean. Proceedings of the National Academy of Sciences,

USA, 96, 15354-15361 (1999).

Rathe, G.M.: Aldehyde oxidase isoenzymes (E.C.1.2.3.1) in potato tubers

(Solanum tuberosum). Plant Cell Physiol., 15, 493-499 (1974).

Sagi, M., R.T. Omarov and S.H. Lips: Xanthine dehydrogenase, nitrate

reductase and aldehyde oxidase in ryegrass as affected by nitrogen

and salinity. Plant Sci., 135, 125-135 (1998).

Sambrook, J., E.F. Fritsch and T. Maniatis: Molecular Cloning: A Laboratory

Manual. 2nd Edn. Cold Spring Harbor Laboratory Publication, New

York (1989).

Sekimoto, H., M. Seo, N. Dohmae, K. Takio, Y. Kamiya and T. Koshiba:

Cloning and molecular characterization of plant aldehyde oxidase. J.

Biol. Chem., 272, 15280-15285 (1997).

Sekimoto, H., M. Seo, N. Kawakami, T. Komano, S. Desloire, S. Liotenberg,

A. Marion-Poll, M. Caboche, Y. Kamiya and T. Koshiba: Molecular

cloning and characterization of aldehyde oxidase in Arabidopsis

thaliana. Plant Cell Physiol., 39, 433-442 (1998).

Seo, M., S. Akaba, T. Oritani, M. Delarue, C. Bellini, M. Caboche and T.

Koshiba: Higher act ivity of an aldehyde oxidase in the auxin-

overproducing superroot1 mutant of Arabidopsis thaliana. Plant

Physiol., 116, 687-693 (1998).

Seo, M., H. Koiwai, S. Akaba, T. Komano, T. Oritani, Y. Kamiya and T.

Koshiba: Abscisic aldehyde oxidase in leaves of Arabidopsis thaliana.

Plant J., 23, 481-488 (2000).

Tilki, Fahrettin: Seed germination of Cistus creticus L. and Cistus laurifolius

L. as influenced by dry-heat, soaking in distilled water and gibberellic

acid. J. Environ. Biol., 29, 193-195 (2008).

Vanlerberghe, G.C. and L. Mclntosh: Mitochondrial electron transport

regulation of nuclear gene expression: Studies with the alternative

oxidase gene of tobacco. Plant Physiol., 105, 867-874 (1994).

Welbaum, G.E., D. Bian, D.R. Hill, R.L. Grayson and M.K. Gunatilaka:

Freezing tolerance, protein composition, and abscisic acid localization

and content of pea epicotyl, shoot, and root tissue in response to

temperature and water stress. J. Exp. Bot., 48, 643-654 (1997).

Zdunek, E. and S.H. Lips: Transport and accumulation rates of abscisic

acid and aldehyde oxidase activity in Pisum sativum L. in response

to suboptimal growth condit ions. J. Exp. Bot . , 52, 1269-1276

(2001).

Zdunek-Zastocka, E., R.T. Omarov, T. Koshiba and H.S. Lips: Activity

and protein level of AO isoforms in pea plants (Pisum sativum L.)

during vegetative development and in response to stress conditions.

J. Exp. Bot., 55, 1361-1369 (2004).

Zdunek-Zastocka E.: Molecular cloning, characterization and expression

analysis of three addehyde oxidase genes from Pisum sativum L.

Plant Physiol. Biochem., 46, 19-28 (2008).

Zeevaart, J.A.D. and R.A. Creelman: Metabolism and physiology of abscisic

acid. Annu. Rev. Plant. Physiol. Plant. Mol. Biol., 39, 439-473 (1988).

Yang et al.98