全 文 ：作物学报 ACTA AGRONOMICA SINICA 2010, 36(12): 2073−2083 http://www.chinacrops.org/zwxb/
ISSN 0496-3490; CODEN TSHPA9 E-mail: xbzw@chinajournal.net.cn

This study was supported by the National Basic Research Program of China (2006CB101700), the National Natural Science Foundation (30971846)
and the Vital Project of Natural Science in Universities of Jiangsu Province, China (09KJA210002).
*
Corresponding author: XU Chen-Wu, E-mail: qtls@yzu.edu.cn, Tel: +86-514-87979358; Fax: +86-514-87996817
Received(收稿日期): 2010-05-18; Accepted(接受日期): 2010-08-01.
DOI: 10.3724/SP.J.1006.2010.02073
Cloning of an ABC1-like Gene ZmABC1-10 and Its Responses to Cadmium and
Other Abiotic Stresses in Maize (Zea mays L.)
GAO Qing-Song, YANG Ze-Feng, ZHOU Yong, ZHANG Dan, YAN Cheng-Hai, LIANG Guo-Hua,
and XU Chen-Wu*
Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology / Key Laboratory of Plant Functional Genomics of the Ministry of Education,
Yangzhou University, Yangzhou 225009, China
Abstract: Cadmium is a non-essential heavy metal that is extremely toxic to plants and animals. Previous studies have shown that
several proteins associated with the Activity of the bc1 complex (ABC1) protein family participate in plant responses to cadmium.
Here we presented the cloning and characterization of an ABC1-like gene, ZmABC1-10, from maize (Zea mays L.). The full-length
2 519 bp cDNA of maize ABC1-10 gene contained an open reading frame (ORF) of 2 250 bp encoding a membrane-binding pro-
tein with a predicted localization in the chloroplast. A promoter scan detected numerous cis-elements implicated in abiotic stress,
light, and phytohormone responses. Expression profile analysis indicated most expression of this gene occurred in green tissues.
Cadmium treatment revealed that expression of this gene could be induced and was correlated with plant development. In addition
to cadmium, ZmABC1-10 expression was also affected by a broad range of abiotic factors, such as ABA, H2O2, drought and dark-
ness. A total of 19 members of maize ABC1 family were identified with the B73 maize genomic sequence. Phylogenetic analysis
using 148 ABC1 proteins from 8 representative species of plant kingdom revealed that divergence occurred and species-specific
expansion contributed to the evolution of this family in plants. Collectively, our data suggest that ZmAbc1-10 is a cadmium-
esponsive factor and may play potential roles in the plant adaption to diverse abiotic stresses.
Keywords: Maize; ABC1-like gene; Cloning; Cadmium response; Abiotic stress
一个玉米类 ABC1基因 ZmABC1-10的克隆及其对镉等非生物胁迫的应答
高清松 杨泽峰 周 勇 张 丹 闫成海 梁国华 徐辰武*
扬州大学江苏省作物遗传生理重点实验室 / 教育部植物功能基因组学重点实验室, 江苏扬州 225009
摘 要: 镉是一种非必需的重金属元素, 对动植物有严重毒害作用。几个与 ABC1(activity of the bc1 complex)家族有
关的基因参与植物镉胁迫的应答。本研究从玉米中克隆并鉴定了一个类 ABC1 基因, 命名为 ZmABC1-10。该基因
cDNA全长 2 519 bp, 包含一个 2 250 bp的开放阅读框, 编码一个预测的叶绿体膜蛋白。启动子顺式元件扫描发现该
基因含有大量的非生物胁迫、光以及植物激素应答元件。表达模式分析表明, 该基因主要在叶片、茎秆等绿色组织
中表达。镉处理实验表明, 该基因能够被诱导并且受植物发育时期的调控。除镉之外, 该基因还受多种非生物因素包
括 ABA、H2O2、干旱和黑暗的共同调控。此外, 本研究利用基因组序列信息共鉴定出 19个玉米 ABC1基因。对植物
界 8个代表性物种中 148个 ABC1蛋白进行系统发育分析表明, 在长期进化过程中植物 ABC1蛋白已经发生了分化;
物种特异性扩张是植物中该家族进化的主要动力。这些结果表明 ZmAbc1-10是一个镉应答因子并且可能在植物对非
生物胁迫的适应中发挥重要作用。
关键词: 玉米; 类 ABC1基因; 克隆; 镉应答; 非生物胁迫
Plant growth and photosynthetic efficiency are greatly
impaired by abiotic factors, such as drought, high salinity,
low temperature and heavy metals. The accumulation of
heavy metals in soil and water has become a potential
risk to modern agriculture. Cadmium (Cd) and its com-
pounds, which are ubiquitous in the environment, disturb
plant life cycles and, therefore, reduce crop yields; they
are also extremely toxic to animals and human when they
2074 作 物 学 报 第 36卷

enter the food chain [1-2]. At the cellular level, Cd inhibits
the photosynthesis of plants by damaging the photosyn-
thetic apparatus and by interfering with pigment synthe-
sis [3]. Cd also affects the plant absorbability of micronu-
trients [3-4], inactivates proteins and enzymes by binding
to sulfhydryl groups and by displacing co-factors of en-
zymes and causes oxidative stress by accumulating reac-
tive oxygen species (ROS) [4-6]. To survive these injuries,
plants have produced a series of cysteine-rich compounds
for the chelation of metal ions and for detoxification,
such as metallothioneins and phytochelatins [7-8]. It has
also been found that different types of transporters, e.g.,
P-type ATPases and ATP-binding cassette (ABC) trans-
porters, participate in plant heavy metal tolerance by
pumping them into vacuoles or apoplasts [9-10]. Moreover,
several direct or indirect signaling pathways, such as
MAPK cascades, jasmonate, ethylene, and H2O2 path-
ways, were found to modulate gene expression in re-
sponse to heavy metals in plants [11-13]. Our understanding
of these mechanisms is growing but still not complete.
Recently, several proteins associated with the ABC1
protein family, such as AtOSA1 in Arabidopsis thaliana [14]
and BjCdR11 in Brassica juncea [15], have been found to
participate in the plant Cd response. The ABC1 (Activity
of bc1 complex) family belongs to protein kinase fami-
lies with two independent origins: a mitochondrial origin
and a chloroplast origin [16]. Studies in yeast, Providencia
stuarti, Escherichia coli, and humans have shown that
proteins of this family in the mitochondria of eukaryotes
and in prokaryotes are essential for ubiquinone (coen-
zyme Q or Q) biosynthesis [17-21], respiratory electron
transfer and antioxidation [22-24]. A chloroplast ABC1
member AtOSA1, however, was found to be implicated
in Cd response and the balance of oxidative stress in
Arabidopsis [14]. Despite sharing homology with the mi-
tochondrial Abc1 of Saccharomyces cerevisiae, this pro-
tein was not able to restore the respiratory defect of yeast
abc1 mutants, indicating that there is a functional differ-
entiation between chloroplast and mitochondrial ABC1s.
In addition, BjCdR11, a homologue of the Arabidopsis
ABC1 gene At3g07700, was also found to be Cd-regu-
lated in a comprehensive cDNA-AFLP analysis in B.
juncea [15].
To investigate potential ABC1-like genes in maize
(Zea mays L.) and their roles under Cd stress, we carried
out a BLAST search in the National Center for Biotech-
nology Information (NCBI) (http://blast.ncbi.nlm.nih.
gov/Blast.cgi) with the coding sequence of Arabidopsis
AtOSA1 (TAIR accession number At5g64940). An in-
complete maize mRNA sequence (GenBank accession
number AY106973), 1 463 bp in length, was found to
share similarity with the query sequence. We then per-
formed a rapid amplification of cDNA ends (RACE) as-
say to acquire the full-length cDNA (FLcDNA) of this
gene, which was designated as ZmABC1-10, according to
the systematic nomenclature of maize ABC1 genes. Se-
quence characteristics and expression patterns were ana-
lyzed to explore the potential functions of the gene. Ad-
ditionally, a phylogenetic analysis spanning five plant
lineages was conducted. To our knowledge, this study is
the first report of ABC1 genes in maize and will lay a
solid foundation for studies of ABC1 genes in plants.
1 Materials and methods
1.1 Plant material, treatments, and RNA isolation
Seeds of maize inbred line A188 were germinated and
cultivated in sand under normal conditions. Trefoil seed-
lings were used for further experiments. For Cd treatment,
seedlings were initially cultivated in hydroponic cultures
with aerated conditions for one week. The components of
the hydroponic culture were as follows: 0.75 mmol L−1
K2SO4; 0.25 mmol L−1 KH2PO4; 0.1 mmol L−1 KCl; 0.6
mmol L−1 MgSO4; 2.0 mmol L−1 Ca(NO3)2; 4.0 μmol L−1
Fe-EDTA; 1.0 μmol L−1 H3BO3; 1.0 μmol L−1 MnSO4;
1.0 μmol L−1 ZnSO4; 0.1 μmol L−1 CuSO4; 5.0×10−3 μmol
L−1 (NH4)6Mo2O4, pH 5.5. Cd concentrations of 0, 0.2,
0.5, 1.0, and 5.0 μmol L−1 were applied in the form of
CdCl2, and the seedlings were cultivated for an additional
two weeks. For Cd treatments of different times, seed-
lings were grown in hydroponic culture in the presence
or absence of 1.0 μmol L−1 CdCl2, and the leaves were
sampled every week until the fifth week. The hydroponic
medium was renewed twice a week.
H2O2 and ABA treatments were conducted by spraying
the seedling leaves and irrigating the roots with 500 μL
L−1 H2O2 and 100 μmol L−1 ABA in 0.2% (V/V) Tween 20,
respectively. Leaves were sampled 6 h later. The seed-
lings were also transferred into a growth chamber at 4℃
with 14 h light/10 h dark cycle for 24 h to test the effect
of low temperature. To estimate the impact of drought
stress, we stopped watering the seedlings until the leaves
were completely rolled. For darkness treatment, the seed-
lings were kept under normal conditions without light for
48 h. The mature leaves, stems, tassels, ears, and seeds of
maize A188 were sampled from well-grown field plants,
and the young roots were sampled from seedlings for
tissue-specific expression analysis.
Total RNA was isolated using the RNAiso Plus re-
agent (TaKaRa, Dalian, China) according to the manu-
facturer’s instructions and treated with DNase I (TaKaRa,
Dalian, China) to remove any of the DNA contamination.
The quality of RNA was checked by UV spectropho-
tometer (Eppendorf, Hamburg, Germany) and 1.0% aga-
rose gel electrophoresis.
1.2 Cloning of ZmABC1-10 FLcDNA
The 5RACE assay was conducted using 5-Full RACE
Kit (TaKaRa, Dalian, China) following the manufac-
turer’s instructions. Gene-specific outer and inner prim-
ers were designed based on the maize mRNA sequence
(GenBank accession number AY106973) using the
Primer Premier 5.0 software (http://www.PremierBio-
第 12期 高清松等: 一个玉米类 ABC1基因 ZmABC1-10的克隆及其对镉等非生物胁迫的应答 2075


soft.com/). Nested PCR was conducted using different
combinations of gene-specific primers and the 5RACE
Outer and Inner primers provided by the kit. PCR products
were purified from the gel using an agarose gel DNA
purification kit (TaKaRa, Dalian, China) and ligated into
pMD 18-T vectors (TaKaRa, Dalian, China) for sequenc-
ing. The primer sequences were listed in Table 1.
For 3RACE, first strand cDNAs were synthesized us-
ing M-MLV Reverse Transcriptase (TaKaRa, Dalian,
China) with a 3RACE Adapter primer. Nested PCR, PCR
product purification, and vector ligation were conducted
as described above. DNA synthesis and sequencing were
performed by Invitrogen Biotechnology Co., Ltd.
(Shanghai, China). Sequence assembly was accom-
plished using the DNAMAN 4.0 software (http://www.
lynnon.com/).
1.3 Bioinformatics analyses
The open reading frame (ORF) of ZmABC1-10 was
predicted using ORF Finder (http://www.ncbi.nlm.nih.
gov/gorf/gorf.html). The genomic sequence of ZmABC1-
10 was retrieved from the B73 maize sequencing data-
base (http://www.maizesequence.org/index.html), while
the gene structure was determined using the gene struc-
ture display server (GSDS) utility (http://gsds.cbi.pku.
edu.cn/index.php)[25]. Basic parameters of ZmAbc1-10
protein were calculated with the ProParam tool (http://
au.expasy.org/tools/protparam.html). The conserved do-
mains and transmembrane helices were predicted using
the InterProScan (http://www.ebi.ac.uk/Tools/InterPro-
Scan/)[26], SMART (http://smart.embl-heidelberg.de/)[27-28],
Pfam (http://pfam.sanger.ac.uk/search?tab=searchSequence
Block)[29], and TMHMM (http://www.cbs.dtu.dk/services/
TMHMM-2.0/)[30] servers.
1.4 Promoter sequence analysis and subcellular
localization
A total of 2 kb of the genomic sequence upstream
from the start codon of ZmABC1-10 gene was submitted
to the plant cis-acting regulatory DNA elements (PLACE)
database (http://www.dna.affrc.go.jp/PLACE/signalscan.
html) for a promoter cis-element scan [31]. The online
TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) [32-33]
and WoLF PSORT (http://wolfpsort.org/)[34] servers were
used for protein subcellular localization prediction.
1.5 Database retrieval and phylogenetic analysis
To identify all the members of maize ABC1 family, we
downloaded the Hidden Markov Model profile of the
ABC1 domain (Pfam accession number PF03109) from
the Pfam database (http://pfam.sanger.ac.uk/), and used
the consensus sequence to retrieve entries from the B73
maize sequencing database as well as the NCBI non-
redundant protein database. All retrieved protein se-
quences were submitted to the Pfam and SMART data-
bases to check the existence of the featured domain.
To illustrate the phylogenetic relationships among
plant ABC1 proteins, we also collected members of other
plant species through multiple database retrieval.
BLASTP searches against the TAIR (The Arabidopsis
Information Resource) database (http://www.arabidopsis.
org/) and the TIGR (The Institute for Genomic Research)
rice annotation database (http://rice.plantbiology.msu.
edu/) were performed to identify all proteins of the
Arabidopsis and rice ABC1 families using the ABC1
consensus sequence as a query. BLASTP searches were
also performed against the DOE Joint Genome Institute
database (http://www.jgi.doe.gov/) to retrieve all ABC1
members in the algous plants Chlamydomonas reihardtii
and Ostreococcus tauri, the moss plant Physcomitella
patens, the fern Selaginella moellendorffii and the di-
cotyledon poplar (Populus trichocarpa). The phyloge-
netic tree was constructed using MEGA 4.0 software [35]
with the neighbor-joining (NJ) method, and the bootstrap
test was carried out with 1000 resamplings.
1.6 RT-PCR and real-time quantitative PCR
analyses
PCR primers were designed based on the cloned
ZmABC1-10 cDNA sequence, the maize Cu/ZnSOD
mRNA sequence (GenBank accession number X17565)
and the maize Actin1 mRNA sequence (GenBank acces-
sion number NM_001155179) using Primer Premier 5.0
software. The maize Actin1 gene was selected as an in-
ternal control as in previous studies [36], and the expres-
sion of maize Cu/ZnSOD was examined as a reference
under oxidative stress. Overall, 1 μg of the total RNA
was used for reverse transcription using the first Strand
cDNA Synthesis Kit (TaKaRa, Dalian, China). cDNAs
were diluted two-fold for RT-PCR analysis. Each reaction
contained cDNA templates, 1×PCR buffer, 0.2 mmol L−1
dNTPs, 0.4 μmol L−1 of both forward and reverse primers
and 0.75 U Taq DNA polymerase (TaKaRa, Dalian,
China). Distilled water was added to a final volume of 25
μL. The PCR conditions included an initial incubation at
95℃ for 5 min, followed by 28 cycles of 94℃for 30 s,
55℃ for 30 s and 72℃ 30 s with a final extension at 72
℃ for 10 min. Twenty microliters of the PCR products
were separated in a 1.0% agarose gel and stained with
ethidium bromide for visualization.
For real-time PCR analysis, the cDNAs were diluted
almost twenty-fold. PCR amplification was performed in
an optical 96-well plate on an ABI PRISM 7500
Real-time PCR Instrument (Applied Biosystems, Foster
City, CA). Each reaction contained 10 μL 2×SYBR Pre-
mix Ex Taq (TaKaRa, Dalian, China), 2.0 μL cDNA tem-
plates, 0.4 μL 50× ROX Reference Dye II and 0.2 μmol
L−1 gene-specific primers in a final volume of 20 μL. The
PCR conditions included an initial incubation at 95℃
for 30 s, followed by 40 cycles of 95℃ for 5 s and 55℃
for 34 s. The specificity of the PCR reactions was deter-
mined by melting curve analyses of the products. Relative
quantification was calculated as
(control sample)target
target
(control sample)ref
ref
( )
( )
CP
CP
E
E
−
−
△
△
[37].
2076 作 物 学 报 第 36卷

Three technical replicates were performed for each sam-
ple, and three biological samples were analyzed for each
treatment. The Student’s t-test was performed as a test of
significance.
2 Results
2.1 cDNA cloning of ZmABC1-10 gene
In this analysis, we cloned one of the maize ABC1-like
genes through a BLAST search and subsequent RACE
assay. As a result, 1 440 bp and 680 bp products from the
5 and 3 ends were amplified, respectively (Fig. 1), with
different primer combinations (Table 1). After sequenc-
ing and splicing, a full-length of 2 519 bp cDNA was
obtained. To avoid any mistakes in this process, we per-
formed a long distance PCR (LD-PCR) to confirm the
new assembly with an amplicon of 2 153 bp. The se-
quencing result of this product was completely consistent
with the cDNA sequence acquired by RACE. The
FLcDNA was then deposited in GenBank with an acces-
sion number of GU228508.



Fig. 1 5 and 3 products of ZmABC1-10 cDNA
The 5′ RACE (A) and 3′ RACE (B) products were analyzed by electro-
phoresis on 1.0% agarose gels.
M: DL5000 (A) and DL2000 (B) DNA markers (TaKaRa, Dalian,
China); 5: 5 RACE products; 3: 3RACE products.

Table 1 Primers used in this study
Type Name Orientation Primer sequence (5–3) Product (bp)
RACE reaction 5RACE Outer Primer Forward CATGGCTACATGCTGACAGCCTA
5RACE Inner Primer Forward CGCGGATCCACAGCCTACTGATGATCAGTCGATG
5specific outer primer Reverse TCCCAGTTCTGCGGTTGCCATTTCTC 1704
5specific inner primer Reverse CAGCAATGTTTCCTGGATGCGGGTCA 1440
3RACE Outer primer Reverse TACCGTCGTTCCACTAGTGA
3specific outer primer Forward CAGAGACTTGCTGCAATCGG 832
3specific inner primer Forward GAGATTGCTAAGCCATATGC 680
RACE validation LD-PCR primers Forward Reverse
GAGCGGGCTCGGATACGACGAT
GCCTATGCTAAATCGTGGTGGG 2153
RT-PCR ZmABC1-10 Forward Reverse
TACAATTTGTTCCGCCAACC
TGCCGATCCAACTTCTTAACCT 299
Maize Cu/ZnSOD Forward Reverse
TGGCCCTACCACTGTCACTG
ATGTTTGCAACACCGTCTGC 213
Real-time PCR ZmABC1-10 Forward Reverse
TTTTCGGGCTGCAAGTTCTGA
CCTATGCTAAATCGTGGTGGG 200
Actin ZmActin1 Forward Reverse
ATGTTGCTATCCAGGCTGTTCT
TTCATTAGGTGGTCGGTGAGGT 175

An ORF that was 2 250 bp in length was identified for
this gene encoding 749 amino acids (Fig. 2), among
which 94 were negatively charged and 109 were posi-
tively charged. The lengths of the 5 and 3 UTRs were 58
and 211 bp, respectively. Calculated molecular weight of
the protein was 84 370 Da with an estimated pI of 9.48.
The grand average of hydropathicity (GRAVY) value
was –0.289, suggesting that the protein was hydrophilic.
Twenty exons with an average length of 112 bp spanned
10 113 bp of the ZmABC1-10 genomic region on chro-
mosome 5 (Fig. 3). In addition, 19 introns covered over
75% of the genomic sequence, which was consistent with
the fact that maize genes contain more large introns
caused by insertion of repetitive elements than do genes
of other grass family species [38-39].
2.2 Promoter sequence analysis
The cis-acting regulatory DNA elements play key
roles in the transcriptional regulation of genes controlling
various biologic processes. To identify putative cis-
elements of ZmABC1-10, we subjected 2 kb of the ge-
nomic sequence upstream from the translation start
codon to the PLACE database for scanning. As a result, a
total of 381 plant cis-elements were identified, among
which 164 elements longer than 6 bp were selected for
further analyses. Results showed that 23 of the 164
cis-elements were implicated in responses to abiotic
stresses (drought, flooding, and low temperature); 23
participated in light signal perception and 11 were re-
sponsive to phytohormones (ABA, Auxin, and GA).
None of them, however, was found to be involved in
heavy metal response.
2.3 Conserved domain prediction and subcellular
localization in silico
To identify the conserved domains of the ZmAbc1-10
第 12期 高清松等: 一个玉米类 ABC1基因 ZmABC1-10的克隆及其对镉等非生物胁迫的应答 2077




Fig. 2 cDNA and deduced amino acid sequences of the ZmABC1-10 gene
Start and stop codons are indicated by single- and triple-asterisks, respectively. Underlined nucleotide sequences represent the 5 and 3 UTRs.
The blank frame indicates a conserved ABC1 domain.



Fig. 3 Schematic structure of the ZmABC1-10 gene
Exons and introns are shown by filled boxes and single lines, respectively. Thick lines at both ends represent UTR sequences. The location of ABC1
domain is shown in black. Intron phases are indicated by the numbers 0, 1, and 2 above the single lines.
2078 作 物 学 报 第 36卷

protein, we scanned the Pfam and SMART databases
with its amino acid sequence. An ABC1 domain (Pfam
accession number PF03109) and a putative STYKc
kinase domain (SMART accession number SM00221)
were detected, located from the 266th to the 386th and
the 278th to the 575th amino acid, respectively (Fig. 4),
suggesting that the protein belongs to the ABC1 family
and has possible kinase activity. However, no featured
domains of ABC transporters were identified. A trans-
membrane helix was found at the C-terminus, which was
suggestive of a membrane-binding protein. Moreover,
online subcellular localization using both the TargetP and
PSORT servers all predicted a chloroplast localization
with high reliability. The length of the presequence was
58 amino acids.



Fig. 4 Schematic illustration of topology of ZmAbc1-10 protein
The white box on the left represents a chloroplast targeting peptide. The
dashed box denotes the ABC1 domain. The black box in the middle
indicates the STYKc domain. The gray barrel on the right represents the
predicted transmembrane span.
2.4 Identification and phylogenetic analysis of
plant ABC1 genes
The new completion of the B73 maize genome [40]
prompted us to identify all the maize ABC1 genes. The
consensus sequence of the ABC1 domain was used to
retrieve entries from the B73 sequencing database and
the NCBI non-redundant protein database. Altogether 19
putative maize ABC1 genes were identified (Table 2),
which were designated as ZmABC1-1 to ZmABC1-19
according to their chromosomal locations. These ABC1
genes were distributed across all maize chromosomes
except for chromosomes 1 and 9 with the largest number
of genes located on chromosome 5 (Table 2).
To explore the evolutionary pattern and phylogenetic
relationships of ABC1 proteins among plant kingdom, we
systematically identified 148 ABC1s from eight repre-
sentative plant species (Table 3). An unrooted tree was
constructed using MEGA 4.0 software with the NJ
method (Fig. 5). The 148 proteins could be divided into
nine subfamilies (A to I) according to their evolutionary
relationships, which harbored 26, 19, 2, 26, 13, 10, 17, 15,
and 20 members, respectively. The members of each
subfamily came from both lower and higher plants except
for subfamily C, indicating that the main characteristics

Table 2 Basic information for ABC1 family genes in maize
Gene a Accession No. b Exon c Chromosome d CDS (bp) e No. of aas f
ZmABC1-1 GRMZM2G087201 6 2 1878 625
ZmABC1-2 GRMZM2G305007 21 2 2112 703
ZmABC1-3 GRMZM2G067520 6 2 1875 624
ZmABC1-4 GRMZM2G113264 12 3 1437 478
ZmABC1-5 GRMZM2G377115 14 4 2076 691
ZmABC1-6 GRMZM2G157369 13 4 2328 775
ZmABC1-7 GRMZM2G040720 16 4 1599 532
ZmABC1-8 NM_001147061 11 5 1530 509
ZmABC1-9 EU975140 6 5 1203 400
ZmABC1-10 GRMZM2G040511/GU228508 20 5 2250 749
ZmABC1-11 GRMZM2G008643 14 5 1575 524
ZmABC1-12 GRMZM2G140917 20 6 2880 959
ZmABC1-13 GRMZM2G020627 7 6 1206 401
ZmABC1-14 NM_001156804 11 7 1656 551
ZmABC1-15 NM_001154617 13 7 2154 717
ZmABC1-16 EU957240 8 8 1836 611
ZmABC1-17 GRMZM2G315125 7 8 2139 712
ZmABC1-18 GRMZM2G091267 12 8 1437 478
ZmABC1-19g GRMZM2G368486 11 10 1596 531
a Systematic nomenclature of maize ABC1 genes. b Accession number of putative ABC1 genes in the B73 maize sequencing database or GenBank.
c The number of exons in putative ZmABC1 genes. d Chromosome on which the ZmABC1 gene is located. e Coding sequence. f The number of protein
amino acids of ZmABC1 genes. g ZmABC1 gene that encodes part of the ABC1 domain.
第 12期 高清松等: 一个玉米类 ABC1基因 ZmABC1-10的克隆及其对镉等非生物胁迫的应答 2079


of these subfamilies were established before the origin of
higher land plants. Subfamily C contained only se-
quences from O. tauri, suggesting that this subfamily had
already existed in algous plants, and divergence probably
occurred after the formation of land plants. This result
also suggested that the algous plant C. reihardtii might
have lost its members of this subfamily over the course
of long-period evolution. Besides the subfamilies, 15
pairs of paralogous genes (1 for O. tauri, 6 for P. patens,
2 for S. moellendorffii, 1 for maize, 3 for poplar, and 2
for Arabidopsis) were noticed on the terminal of the
phylogenetic tree, illustrating that species-specific ex-
pansion contributed to the evolution of this family in
these species. The ZmAbc1-10 protein was grouped in
subfamily D with the Arabidopsis At5g64940 and
At3g07700 proteins, indicating that they might share the
same functions. The closest homologue is Os02g36570,
which shared 83% amino acid identities with
ZmAbc1-10. In addition, the ZmAbc1-9 and ZmAbc1-7
proteins were also found to be clustered in this subfamily.
2.5 Tissue-specific expression of ZmABC1-10 gene
We investigated the tissue-specific expression of
ZmABC1-10 through real-time PCR analysis. Results
showed that the gene was expressed primarily in green
tissues such as leaves and stems. It was also expressed
slightly in tassels and ears but had almost no expression
in young roots and immature seeds (Fig. 6-A). The real-
time PCR results were then confirmed by semi-quantita-
tive RT-PCR (Fig. 6-B).

Table 3 Number of ABC1 genes identified from eight representa-
tive plant species
Lineage Species Gene number
Algae Chlamydomonas reihardtii 14
Ostreococcus tauri 19
Moss Physcomitella patens 22
Fern Selaginella moellendorffii 19
Monocots Oryza sativa 15
Zea mays 19
Dicots Populus trichocarpa 23
Arabidopsis thaliana 17
Total 148





Fig. 5 Phylogenetic tree of the plant ABC1 proteins
Sequences aligned were from eight representative plant species: the algae Chlamydomonas reihardtii and Ostreococcus tauri, the moss plant Phy-
scomitella patens, the fern Selaginella moellendorffii, the dicots poplar and Arabidopsis as well as the monocots rice and maize. The unrooted tree
was constructed using MEGA 4.0 software with the NJ method. Bootstrap testing was performed with 1000 resamplings.
2080 作 物 学 报 第 36卷

2.6 Effect of Cd on ZmABC1-10 expression
To study the differential expression of ZmABC1-10
under Cd stress, we performed Cd treatments for diffe-
rent concentrations and times. Though it does not take
part directly in the cellular redox reactions, Cd can im-
pose oxidative injuries on plants via the inhibition of the
key enzymes of the ROS scavenging systems [1]. The
maize Cu/ZnSOD gene, which encodes one of the major
anti-oxidative enzymes in plants, was used here as a ref-
erence gene. Results showed that ZmABC1-10 expression
was significantly up-regulated by 0.2 μmol L−1 CdCl2 but
became stable with the elevation of Cd concentrations
(Fig. 7-A). The expression of maize Cu/ZnSOD, however,



Fig. 6 Tissue-specific expression of the ZmABC1-10 gene
A: real-time PCR results of the expression profile of ZmABC1-10;
B: confirmation of real-time results by semi-quantitative RT-PCR.
R: young roots; St: stems; L: leaves; T: tassels; E: ears; Se: seeds.



Fig. 7 RT-PCR analyses of ZmABC1-10 expression under Cd
stress
A: expression of ZmABC1-10 under Cd treatment at different concen-
trations; B: time-dependent expression of ZmABC1-10 in the presence
(+) or absence (−) of 1 μmol L−1 CdCl2.
The maize Cu/ZnSOD gene was used as a reference gene under oxida-
tive stress.
was down-regulated by 0.2 μmol L−1 CdCl2, though it
could be induced by higher Cd concentrations (Fig. 7-A).
In addition, expression was found to be suppressed by 5
μmol L−1 CdCl2, suggesting that excessive Cd strongly
reduces the transcription of Cu/ZnSOD [41-42].
Simultaneously, a time-course experiment was carried
out in a hydroponic culture containing 1 μmol L−1 CdCl2.
The seedlings without Cd treatment were used as a con-
trol. In the absence of Cd, expression of both ZmABC1-
10 and Cu/ZnSOD changed as the plant developed (Fig.
7-B). When Cd was added, ZmABC1-10 expression was
first up-regulated at day 14 compared to the control, then
became stable until day 21 and was finally down-regu-
lated at day 28. In contrast, expression of maize Cu/
ZnSOD was continuously up-regulated by 1 μmol L−1
CdCl2 (Fig. 7-B), indicating that durative Cd treatment
imposed oxidative stress on the seedlings.
2.7 Effects of other abiotic stresses on ZmABC1-
10 expression
One of the effects of Cd stress is the induction of wa-
ter deficiency in plant cells which is believed to be
modulated by ABA [15,43], and Cd is also believed to in-
terfere with cellular signaling mediated by H2O2 [1,13]. We
treated the seedlings with ABA, H2O2, drought, and low
temperature (4 ) to test the effects of these abiotic fa℃ c-
tors. Real-time PCR results showed that the expression of
ZmABC1-10 was down-regulated 58% (P<0.01) by 6 h of
ABA treatment (Fig. 8). It could also be down-regulated
by drought (73%, P<0.01). H2O2 is one of the major ROS
and is an important signaling molecule implicated in the
balance of oxidative stress [44-45]. Expression of ZmABC1-
10 was down-regulated 26% (P<0.01) by 6 h of H2O2
treatment (Fig. 8), suggesting the involvement of this
gene in the oxidative response. Though a low-tempera-
ture-responsive element was detected in the gene pro-
moter, expression was not significantly changed by 24 h
of 4 treatment (℃ P>0.05, Fig. 8). Taking into considera-
tion that the light-responsive elements presented in the
promoter and the protein product was predicted to be
localized in chloroplast, we also tested the effect of light



Fig. 8 Real-time PCR results of ZmABC1-10 expression under
diverse abiotic stresses
第 12期 高清松等: 一个玉米类 ABC1基因 ZmABC1-10的克隆及其对镉等非生物胁迫的应答 2081


on ZmABC1-10 expression. Results showed that the ex-
pression was down-regulated 74% (P<0.01) by 48 h of
darkness treatment (Fig. 8).
3 Discussion
Cd is one of the most important environmental pol-
lutants which can be easily taken up through transporters
of root epidermal cells and transferred to aerial parts,
causing injuries to plant cells. In this analysis, we cloned
one of the maize ABC1-like genes and examined its ex-
pression under Cd stress. Treatment of different concen-
trations revealed that the gene expression could be in-
duced, but expression did not seem to be dose-dependent
due to the fact that we only used a few Cd concentrations.
Similarly, treatment for different lengths of time also
revealed an up-regulation of ZmABC1-10 expression by
Cd. These results suggested that the gene is Cd-regulated.
However, it is worth emphasizing that ABC1 proteins are
not related to the ABC transporters because they do not
harbor any featured domains of ABC transporters such as
nucleotide-binding domains. The response of ZmABC1-
10 to Cd likely does not occur by the direct trafficking of
Cd2+ by its protein product. Furthermore, ZmAbc1-10 is
also likely not a direct protein of heavy metal binding in
vivo because this protein lacks cysteines and does not
harbor any known metal-binding motifs such as C-X-C,
C-C, and C-X-X-C motifs of the metallothioneins [7].
In the cDNA-AFLP analysis conducted by Fusco et
al.[15], four genes induced by Cd in B. juncea were
homologous to Arabidopsis MYB, GBF, TGA, and Zinc-
finger transcription factors (TFs) that were implicated in
extensive stress responses. Several other TFs that re-
sponded to Cd, such as DREB2A [46] and WRKY53 [47],
were also found to be modulated by drought, cold and
salt stresses. Thus, it was suggested that TFs responding
to heavy metals shared signaling pathways with other
stress-responsive TFs [1]. In this analysis, expression of
ZmABC1-10 was found to be modulated by a broad range
of abiotic factors, supporting the inference above; i.e., Cd
shared common signaling pathways with other abiotic
stresses.
The promoter of ZmABC1-10 possessed diverse
cis-elements implicated in responses to abiotic stresses,
light and phytohormones. Interestingly, the degree at
which ZmABC1-10 was down-regulated by ABA treat-
ment (58%) was greater than that by H2O2 stress (26%),
though ABA imposed H2O2 stress in an indirect way [48].
This result indicated that the gene might be involved in
an ABA-dependent signaling besides the H2O2 pathway.
Osmotic stress caused by drought and cold can activate
common signaling pathways in plants, such as the cal-
cium-dependent protein kinase pathway [49-50]. The ex-
pression of ZmABC1-10 was remarkably decreased by
drought (73%) but was not affected by low temperature,
suggesting the existence of independent pathways of cold
stress response [51]. In addition, ZmAbc1-10 was likely
not an upstream component of the signaling pathways
because the gene expression was down-regulated by most
of the abiotic factors examined.
The consistent theme concerning roles of ABC1 pro-
teins in several species (yeast, P. stuarti, E. coli, human
beings, etc.) is the regulation of ubiquinone biosynthe-
sis[17-21], which acts as an electron carrier in the respira-
tory electron transfer chain and as a lipid-soluble anti-
oxidant. It is not clear whether ABC1 proteins in the
chloroplasts of higher plants can perform similar func-
tions. The Arabidopsis genome encodes 17 ABC1 pro-
teins, four of which were isolated from the plastoglobules
of the chloroplast (TAIR accession numbers At5g05200,
At1g79600, At1g71810, and At4g31390) but were also
suggested to be involved in quinone biosynthesis [52].
Considering the existence of an electron transfer chain in
chloroplasts similar to that in mitochondria, which con-
sists of the PS II, cytochrome b6f and PS I complexes, it
is easily conceivable that chloroplast ABC1 proteins par-
ticipate in photosynthetic electron transfer by regulating
quinone (plastoquinone for example) metabolism. Study
of the electron transport of the atosa1 mutant, however,
revealed no significant difference between the mutant
and wild type [14], adding additional complexity to the
roles of chloroplast ABC1 proteins. Nonetheless, we still
cannot exclude these proteins from having roles in pho-
tosynthesis because numerous light-responsive cis-ele-
ments were identified in the promoter of ZmABC1-10,
and the gene expression was remarkably affected by
darkness treatment.
Abiotic stresses are major restrictions to crop produc-
tivity[53]. Studies of the mechanisms by which plants cope
with adverse environments will be of potential value to-
ward crop breeding to improve production. Our results
provided evidence that ZmABC1-10 was responsive to
extensive abiotic factors including Cd and likely plays a
role in the plant environmental adaption. In addition,
quite a few ABC1s were identified from different plant
lineages, suggesting that this family might be crucial in
the life cycles of plants. Phylogenetic analysis of these
proteins revealed nine distinct subfamilies, indicating
that divergence probably occurred during the long evolu-
tion period of this family.
4 Conclusions
An ABC1-like gene, ZmABC1-10, was cloned from
maize inbred line A188 via database search as well as
RACE assay. This gene encoded a predicted chloroplast
protein and expressed mainly in the green tissues. The
gene expression was responsive to Cd and was correlated
with plant development. Besides Cd, expression of
ZmABC1-10 could also be modulated by a broad range of
abiotic factors such as ABA, H2O2, drought, and darkness.
These results illustrated that, unlike yeast and prokaryotic
2082 作 物 学 报 第 36卷

ABC1 proteins, ZmAbc1-10 may play potential roles in
the maize responses to Cd and other abiotic stresses.
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