CCCH-Type Zinc Finger Family in Maize: Genome-Wide
Identification, Classification and Expression Profiling
under Abscisic Acid and Drought Treatments
Xiaojian Peng., Yang Zhao., Jiangang Cao, Wei Zhang, Haiyang Jiang, Xiaoyu Li, Qing Ma, Suwen Zhu,
Key Laboratory of Crop Biology of Anhui Province, Anhui Agricultural University, Hefei, China
Background: CCCH-type zinc finger proteins comprise a large protein family. Increasing evidence suggests that members of
this family are RNA-binding proteins with regulatory functions in mRNA processing. Compared with those in animals,
functions of CCCH-type zinc finger proteins involved in plant growth and development are poorly understood.
Methodology/Principal Findings: Here, we performed a genome-wide survey of CCCH-type zinc finger genes in maize (Zea
mays L.) by describing the gene structure, phylogenetic relationships and chromosomal location of each family member.
Promoter sequences and expression profiles of putative stress-responsive members were also investigated. A total of
68 CCCH genes (ZmC3H1-68) were identified in maize and divided into seven groups by phylogenetic analysis. These 68
genes were found to be unevenly distributed on 10 chromosomes with 15 segmental duplication events, suggesting that
segmental duplication played a major role in expansion of the maize CCCH family. The Ka/Ks ratios suggested that the
duplicated genes of the CCCH family mainly experienced purifying selection with limited functional divergence after
duplication events. Twelve maize CCCH genes grouped with other known stress-responsive genes from Arabidopsis were
found to contain putative stress-responsive cis-elements in their promoter regions. Seven of these genes chosen for further
quantitative real-time PCR analysis showed differential expression patterns among five representative maize tissues and
over time in response to abscisic acid and drought treatments.
Conclusions: The results presented in this study provide basic information on maize CCCH proteins and form the
foundation for future functional studies of these proteins, especially for those members of which may play important roles
in response to abiotic stresses.
Citation: Peng X, Zhao Y, Cao J, Zhang W, Jiang H, et al. (2012) CCCH-Type Zinc Finger Family in Maize: Genome-Wide Identification, Classification and Expression
Profiling under Abscisic Acid and Drought Treatments. PLoS ONE 7(7): e40120. doi:10.1371/journal.pone.0040120
Editor: Pierre-Antoine Defossez, Universite ´ Paris-Diderot, France
Received February 14, 2012; Accepted June 1, 2012; Published July 6, 2012
Copyright: ? 2012 Peng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 31071423 and 11075001). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Zinc finger motifs in proteins consist of cysteines and/or
histidines which coordinate a zinc ion to form local peptide
structures that are required for specific biological functions .
Based on their structural and functional diversities, zinc finger
proteins have been categorized into at least 14 families, such as
ERF, WRKY, DOF and RING-finger families [2,3,4,5]. CCCH-
type zinc finger proteins, one group of these zinc finger families,
are shown to contain tandem zinc-binding motifs characterized by
three cysteines followed by one histidine [6,7]. A typical CCCH
protein usually contains 1–6 CCCH-type zinc finger motifs. Based
on the different numbers of amino acid spacers between cysteines
and histidines in the CCCH motif, a consensus sequence for these
motifs was defined as C-X4–15-C-X4–6-C-X3-H (X represents any
amino acid) based on the whole-genome analysis of rice and
Arabidopsis CCCH proteins .
CCCH-type zinc finger proteins are RNA-binding proteins by
virtue of the ability of their defining motif to directly bind to RNA,
whereas most of the other zinc finger families are confirmed as
DNA-binding or protein-binding proteins . Although increasing
evidence have demonstrated that CCCH zinc finger proteins
possess RNA binding activity, the precise role of this zinc finger
motif is poorly understood. CCCH motif-containing proteins
comprise a large protein family, and are widely distributed across
eukaryotes. In animals, tristetraprolin (TTP) is a well-character-
ized CCCH zinc finger protein, which contains two such motifs
and binds to AU-rich elements in the 39-untranslated region of
their target transcripts, in most cases mediating mRNA degrada-
tion [10,11]. Another CCCH-type zinc finger protein, zinc-finger
antiviral protein (ZAP) isolated from Rat2 fibroblasts, can directly
bind to specific viral RNA sequences through its CCCH zinc
finger motifs and inhibit retroviral RNA production . Yet
other CCCH proteins can control the translation of their target
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mRNAs. For example, the Drosophila protein ZC3H3 regulates
mRNA nuclear adenylation and export .
In Arabidopsis, HUA1, a CCCH-type zinc finger protein with six
tandem CCCH motifs, has been identified as an RNA-binding
protein and likely participates in a new regulatory mechanism for
flower development. This protein is able to associate with
AGAMOUS mRNA so that it can indirectly determine organ
identity specification . AtCPSF30, the Arabidopsis ortholog of
the 30-kD subunit of the cleavage and polyadenylation specificity
factor, was shown to be a nuclear-localized RNA-binding protein
that binds calmodulin . In addition, some CCCH zinc finger
proteins have been demonstrated to be involved in abiotic and
biotic stresses. For example, salt stress-inducible zinc finger protein
1 (AtSZF1) and AtSZF2 were found to be involved in modulating
the tolerance of Arabidopsis plants to salt stress . Recently,
GHZFP1, a CCCH-type zinc finger protein from cotton, was
revealed to enhance tolerance to salt stress and resistance to fungal
disease in transgenic tobacco . Although the majority of the
CCCH motifs bind RNA, DNA binding CCCH-type zinc finger
proteins have also been confirmed. PEI1, an embryo-specific
transcription factor in Arabidopsis, has been shown to play an
important role during embryogenesis .
Although CCCH-type zinc finger proteins are known to have
important roles in various aspects of plant growth and de-
velopment, their functions remain poorly characterized in maize.
Recently, the maize genome was completed with high quality
sequences , which provided an opportunity to perform
a genome-wide analysis of the CCCH gene family in order to
understand the evolutionary history and functional mechanisms of
its members in such an important species. In this study,
a comprehensive analysis of the CCCH gene family was
performed by searching the entire maize genome. In addition,
we investigated the expression patterns of seven potential stress-
responsive genes in five representative tissues and their responses
to abscisic acid (ABA) and drought treatments. The results provide
an important foundation for future cloning and functional studies
of CCCH proteins in maize.
Identification of CCCH Proteins in Maize
After extensive searches of the maize genome database by using
previously reported Arabidopsis and rice CCCH proteins as
BLASTP queries, a total of 68 CCCH genes (designated ZmC3H1
to ZmC3H68) were identified in maize and listed in Table S1. Our
results showed that the maize and Arabidopsis genomes encode the
same number of CCCH proteins, which was similar to that in rice
(67 members). Like Arabidopsis and rice CCCH proteins, the zinc
finger motif in the maize CCCH zinc finger family is highly
conserved. All identified CCCH genes encode proteins varying
from 160 to 1200 amino acids, with a few exceptionally longer or
smaller proteins (Table S1). However, some novel patterns and
features of the maize CCCH zinc finger motif that differ from
other plants were also found. A total of 180 CCCH zinc finger
motifs were recognized by Pfam and SMART among the 68 maize
CCCH proteins, which was greater than those found in Arabidopsis
(152) and rice (150), even though similar numbers of CCCH
proteins were identified in the three plants (Fig. 1A).
Previous studies have indicated that CCCH proteins have
between one and six CCCH-type zinc finger motifs characterized
by three cysteines followed by one histidine. However, our analysis
detected a novel maize CCCH protein (ZmC3H3), which contains
seven copies of the CCCH zinc finger motifs. Therefore, we
divided the maize CCCH proteins into seven groups according to
the number of copies of CCCH motif in each protein. As shown in
Fig. 1B, approximately 59% of all identified maize CCCH
proteins have one or two CCCH motifs, while 17.6% contain
three copies and 16% have five copies. Similar trends were also
found in Arabidopsis and rice (Fig. 1B). After whole-genome analysis
of CCCH proteins in Arabidopsis and rice, the CCCH zinc finger
proteins were re-defined as C-X4–15-C-X4–6-C-X3-H by Wang et
al. (2008) based on the different number of amino acid spacers
between cysteines and histidines, which were originally defined as
C-X6–14-C-X4–5-C-X3-H. In this study, a novel zinc finger motif,
C-X17-C-X6-C-X3-H, was identified in ZmC3H17. Similar to that
reported in Arabidopsis and rice, the most abundant CCCH zinc
finger motifs were found to be C-X8-C-X5-C-X3-H (54.4%) and
C-X7-C-X5-C-X3-H (25%) in the maize CCCH family. It is worth
noting that C-X7-C-X4-C-X3-H, C-X5-C-X4-C-X3-H, C-X7-C-
X6-C-X3-H and C-X8-C-X4-C-X3-H are the largest groups of
motifs among the 20.6% uncommon CCCH zinc finger motifs
(Fig. 1C). Detailed information on the maize CCCH zinc finger
family can be found in Table S2.
To explore sequence characteristics of the most common zinc
finger motifs in the maize CCCH gene family, we performed
sequence alignments of 45 C-X7-C-X5-C-X3-H motifs and 98 C-
X8-C-X5-C-X3-H motifs to generate sequence logos, which were
then compared with the corresponding CCCH motif sequence
logos in rice and Arabidopsis. Moreover, a combined sequence logo
for the two types of motifs was also created using the same method.
The results confirmed that the CCCH zinc finger motif is highly
conserved in the CCCH zinc finger family. As shown in Fig. 2,
there was very little difference among the sequence logos derived
from the three plants. Each logo of the three types of motifs was
similar to the Pfam or SMART sequence logos for CCCH zinc
finger motifs, especially at the four amino acids, three cysteines
and one histidine, which are completely conserved among all the
CCCH motifs. Differences were only found in the combined
sequence logos for C-X8-C-X5-C-X3-H and C-X7-C-X5-C-X3-H
motifs of the three plants. For example, tyrosine was more
conserved than phenylalanine at position C1+3 in both of the
maize and rice combined logos, while the reverse case was
observed in the Arabidopsis combined logo (Fig. 2C).
Phylogenetic and Structural Analysis of Maize CCCH Zinc
To detect the evolutionary relationships within the maize
CCCH zinc finger family, a neighbor-joining (N-J) tree was
constructed based on the alignment of the full-length sequences of
the 68 maize CCCH proteins. According to the phylogenetic
analysis, maize CCCH proteins were divided into seven groups
(group I to group VII) (bootstrap values .50%). Sixty-eight maize
CCCH genes formed 24 sister pairs, and 18 of them showed high
bootstrap support (99%). We also noted that while the majority of
the phylogenetic clades had well-supported bootstrap values, the
phylogenetic relationships of some proteins were unclear and the
bootstrap values were low at the nodes (Fig. 3A). To support the
phylogenetic reconstruction, we executed an exon-intron analysis
by comparing the predicted coding sequence (CDS) with the
genomic sequence of the maize CCCH genes (Fig. 3B). Consistent
with the phylogenetic analysis, genes clustered in the same group
displayed the similar exon-intron structures, especially in the
number of introns, although exceptions to this observation were
also found. For example, ZmC3H35, -30 and -27 in group VII
contain multiple introns, while ZmC3H42, -52, -11, -40 and -15 do
not contain any introns. Moreover, the intron length is also highly
variable, ranging from several tens to approximately 16,000 bases.
Those observations reflect the high sequence diversity of the
CCCH-Type Zinc Finger Family in Maize
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CCCH zinc finger family. Although the sequences between
adjacent cysteines of the zinc finger motif in the CCCH gene
family are highly conserved, the number of motifs encoded by
each protein and spacing sequences between tandem CCCH zinc
finger motifs are diverse.
Besides containing 1–7 copies of CCCH zinc fingers, some
CCCH proteins also carry several other known functional
domains, including ANK, WD-40, KH, RRM, ZF-U1, HELICc,
DEXDc and ZF-Ring (Fig. 4). It is worth mentioning that among
the 68 members of the maize CCCH family, 13 CCCH proteins
contain the RRM domain, and 5 members contain the KH
domain. RRM and KH domains are common RNA-binding
domains in eukaryotes [20,21]. Moreover, RRM and KH
domain-containing proteins have been demonstrated to play
essential roles in many aspects of RNA metabolism, suggesting
that the 18 CCCH proteins harboring these domains may
function as RNA-binding proteins and are involved in RNA
processing. We also noted that the majority of the CCCH
proteins in the same phylogenetic subfamily displayed a similar
domain architecture. For example, all of the group I proteins
contain five copies of the CCCH zinc finger domain except for
ZmC3H20, -49 and -3. In particular, subfamily-specific domains
were also found among the CCCH proteins. For example, the
group II CCCH proteins, including ZmC3H6, -67, -26, -37 and -
60, have a conserved KH domain, suggesting that the KH
domain may play important subfamily-specific functions. Thus,
the phylogenetic reconstruction was further supported by analysis
of the domain architecture.
With the development of comparative genomics, it is possible to
analyze the same protein families among different species. To
evaluate the evolutionary relationships of CCCH genes in maize,
rice and Arabidopsis, a combined N-J tree was constructed from
alignments of the complete sequences of all 203 CCCH proteins
from the three species. We divided the 203 members into 26
groups, designated from A to Z, according to the phylogenetic
clades with at least 50% bootstrap support. The phylogenetic
analysis showed that most maize CCCH genes clustered with their
rice or Arabidopsis counterparts with high bootstrap support (Fig. 5).
Furthermore, we noted that ZmC3Hs were more closely grouped
with OsC3Hs than with AtC3Hs. For instance, ortholog pairs of
maize and rice CCCH proteins were more prevalent in the tree,
which indicated that some ancestor CCCH genes have been
existed before the divergence of maize and rice. The phylogenetic
analysis confirmed the close relationship of maize and rice,
consistent with the evolutionary relationships among the three
species. Generally speaking, the monocot CCCH genes formed
Figure 1. Characterization of CCCH-type zinc finger proteins. (A) Numbers of CCCH proteins and CCCH zinc finger motifs identified in maize,
rice and Arabidopsis. (B) Numbers of CCCH proteins containing 1, 2, 3, 4, 5, 6 or 7 CCCH zinc finger motifs in maize, rice and Arabidopsis. (C) Numbers
of each type of CCCH zinc finger motifs in maize, rice and Arabidopsis.
CCCH-Type Zinc Finger Family in Maize
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sister pairs with the nearest monocot orthologs, while the dicot
CCCH genes formed sister pairs with the nearest dicot orthologs.
However, sister pairs formed by Arabidopsis and maize/rice
orthologs with very strong bootstrap support were also found in
our phylogenetic tree, such as AtC3H7-ZmC3H59 and AtC3H22-
OsC3H18, Notably, while some groups in the phylogenetic tree
consisted of a few CCCH genes of maize, rice and Arabidopsis,
several groups were significantly expanded. For example, group T
contained 32 CCCH members with well-supported bootstrap
values. Generally, genes clustered into the same group of
phylogenetic tree often share similar functional features. A
previous study showed that the Arabidopsis CCCH genes clustered
in the group T in our phylogenetic tree are involved in responses
to abiotic stress, suggesting that maize CCCH genes of this group
may play essential roles in plant stress responses . CCCH genes
with unclear phylogenetic relationships were also found in the
combined tree (Fig. 5).
Chromosomal Locations and Gene Duplication
Based on the starting position of each gene on the chromo-
somes, the 68 maize CCCH genes were found to be unevenly
distributed on chromosomes 1 to 10 (Fig. 6). Chromosome 8
contained the largest number of maize CCCH genes (12) followed
by chromosome 3 (10). By contrast, chromosomes 7 and 9
contained the least number of maize CCCH genes (4). Eight
CCCH genes were located on chromosome 6, six on chromo-
somes 5, 2 and 4, seven on chromosome 10 and five on
Based on the phylogenetic analysis and the chromosomal
locations of the ZmC3H genes, 15 of the 24 sister pairs were
located on the same duplicated chromosomal blocks as previously
reported . On the other hand, a total of 30 maize CCCH
genes were involved in the segmental duplications (Fig. 6). The
highest frequency of segmental duplication events occurred
between chromosomes 8 and 3, which showed six segmental
duplication events, while no duplication events occurred among
the four CCCH genes on chromosome 7. In addition, two gene
clusters (ZmC3H13/14 and ZmC3H46/47) were detected on
chromosomes 8 and 3. Gene clusters may develop as a result of
the birth and death process of tandem duplication, indicating
that genes in the two clusters were involved in tandem
duplication, consistent with the definition of tandem duplication
detection in this study. Interestingly, the four tandem duplicated
genes were also mapped on the same duplicated chromosomal
To explore different selective constrains on duplicated CCCH
genes, the Ks and Ka/Ks ratio for each pair of duplicated
CCCH genes were calculated. Generally, a Ka/Ks ratio .1
indicates accelerated evolution with positive selection, a ratio =1
indicates neutral selection, while a ration ,1 indicates negative
or purifying selection. In this study, all of the 17 duplicated pairs
in the maize CCCH family were investigated. The results showed
that the Ka/Ks ratios for 15 duplicated pairs were ,1, with
most of them being even less than 0.4, suggesting strong
purifying selection (Table 1). However, the other two duplicated
pairs seemed to be under positive selection, as their Ka/Ks ratios
were .1. These results suggested that functions of the duplicated
genes did not diverge much along with the genome evolution
after the duplication events. Based on the Ka/Ks analyses, we
concluded that purifying selection may be largely responsible for
maintenance of function in the maize CCCH family. We also
calculated the duplication dates by calculating the synonymous
substitutions between the 17 duplicated CCCH gene pairs. On
the basis of a substitution rate of 6.561029substitutions per site
per year [23,24], the duplication events for the 15 segmental
duplications were estimated to have occurred approximately
between 5 to 28 million years ago (Mya). However, the
duplication times for the two tandem pairs of ZmC3H13/14
and ZmC3H46/47 were 62.92 and 74.23 Mya, respectively
(Table 1). The results suggested that the two tandem duplication
events occurred before the formation of the segmental duplica-
tion pairs ZmC3H46/14 and ZmC3H47/13.
Figure 2. Sequence logos for common CCCH zinc finger motifs.
(A) C-X7-C-X5-C-X3-H motifs of maize, rice and Arabidopsis. (B) C-X8-C-
X5-C-X3-H motifs of maize, rice and Arabidopsis. (C) C-X7-C-X5-C-X3-H
and C-X8-C-X5-C-X3-H motifs of maize, rice and Arabidopsis.
CCCH-Type Zinc Finger Family in Maize
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Analysis of the Promoters and Sequence Characteristics
of Potential Abiotic Stress-responsive CCCH Genes
As mentioned above, the Arabidopsis genes clustered in the group
T were shown to be involved in response to abiotic stress. The
phylogenetic analysis indicated that group T contains 12 maize
CCCH genes and that are closely related to the Arabidopsis stress-
responsive genes (Fig. 5). This observation prompted us to
investigate possible stress-responsive cis-elements in the promoter
regions of the 12 maize CCCH genes by searching against the
PLACE database. Two types of cis-elements, including the ABA
responsive element (ABRE) and dehydration-responsive element
(DRE), were detected in the current study [25,26]. The results
showed that each of the 12 CCCH genes contain ABRE or DRE
in their 2,000 bp promoter sequences, which may be responsive
for their stress responsiveness (Fig. 7). Although the phylogenetic
analysis showed close relationships of the 12 stress-responsive
genes, we found surprising differences in the numbers of the two
cis-elements in their promoter regions. For example, the promoter
regions of ZmC3H10 and -43 contain multiple putative ABREs
and DREs. In contrast, only two DREs were detected in the
promoter of ZmC3H34. Moreover, we also found that the two cis-
elements are not conserved in the promoter regions of the three
sister pairs involved in segmental duplication (ZmC3H12/51,
ZmC3H39/53 and ZmC3H4/28), consistent with the previous
finding that cis-elements of segmental duplication genes in
Figure 3. Phylogenetic relationship and exon-intron structure of maize CCCH proteins. (A) The unrooted tree was constructed using
MEGA4.0 by the N-J method. Bootstrap values (above 50%) from 1,000 replicates are indicated at each node. (B) Exons and introns are indicated by
white rectangles and thin lines, respectively. The untranslated regions (UTRs) are indicated by thick lines.
CCCH-Type Zinc Finger Family in Maize
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Arabidopsis are less conserved when compared to tandem duplica-
tion genes . Although this observation indicated that the three
segmental duplication pairs of maize CCCH family might not
have some similar regulatory features, each of the segmental
duplication genes contain at least one ABRE element in their
promoter regions, which is a central cis-element of ABA signal
transduction in response to stress treatments . Thus, we
concluded that the duplicated genes may share similar regulatory
pathway in some respects. Notably, while most of the genes
contain both types of stress-responsive elements in their promoter
sequences, only one type of stress-responsive element was detected
in the promoter regions of ZmC3H4 and -34.
To our knowledge, only four CCCH-type zinc finger proteins
(CaKR1, GhZFP1, AtSZF1 and AtSZF2) that confer tolerance to
Figure 4. Schematic structures of maize CCCH proteins. Schematic structures of 68 CCCH zinc finger proteins identified in maize are shown
with names of all the members on the left side of the figure. Different domains are indicated by different boxes denoted at the right bottom corner.
Domain abbreviations: ZF-CCCH, CCCH-type zinc finger domain; ANK, ankyrin repeat domain; WD-40, WD-40 repeat domain; KH, K homology domain;
RRM, RNA recognition motif; ZF-U1, U1-like zinc finger domain; HELICc, helicase superfamily C-terminal domain; DEXDc, DEAD-like helicases
superfamily domain; ZF-Ring, Ring-type zinc finger domain. The proteins were grouped manually according to the number of CCCH zinc finger and
the distribution of other conserved domains.
CCCH-Type Zinc Finger Family in Maize
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stresses in transgenic plants have been isolated [16,17,29]. Thus,
an additional N-J tree was built for the stress-responsive members
using the full-length amino acid sequences. The phylogenetic tree
categorized the 25 stress-responsive proteins into two distinct
subfamilies (I and II) with very strong bootstrap support (Fig. 8A).
Subfamily I contained 13 members, and subfamily II contained 12
members. Sequence alignments showed that each of the
25 CCCH proteins contain two putative CCCH zinc finger
motifs of C-X7–13-C-X5-C-X3-H and C-X5-C-X4-C-X3-H (only
one CCCH motif was detected in ZmC3H39, -53 and -54 by Pfam
or SMART) (Fig. 8B). It should be noted that all of the C-X5-C-
X4-C-X3-H motif-containing proteins of the maize and Arabidopsis
CCCH families clustered into the same phylogenetic clade.
Moreover, the C-X5-C-X4-C-X3-H motif contained in subfamily
I proteins were found to be highly conserved, suggesting the
crucial roles of this motif in subfamily-specific functions. Among
the 13 CCCH genes of subfamily I, 10 genes with ankyrin (ANK)
repeat motifs were identified (Fig. 8C). The ANK repeat motif has
been shown to mediate protein-protein interactions, and it is found
in numerous proteins with diverse functions, such as signal
transduction, transcriptional control and cell cycle regulation
[30,31,32]. These observations prompted us to further analyze the
subfamily I CCCH genes.
Expression Levels of Maize CCCH Genes in Various
Tissues and in Response to Abiotic Stress
Analyses of the sequences and evolutionary relationships
indicated that seven maize CCCH genes were clustered in the
subfamily I, including ZmC3H4, -28, -63, -10, -34, -43 and -54,
which may be a novel subfamily involved in abiotic stress
responses (Fig. 8A). Thus, we used quantitative real-time PCR
(qRT-PCR) to survey the relative expression levels of these seven
members in five representative tissues. The results showed that the
seven genes exhibit differential expression patterns in the five
tissues except for ZmC3H34 and -43 (Fig. 9). Five of the genes,
ZmC3H4, -28, -10, -34 and -43, showed high expression levels in
ears and stems, while ZmC3H54 and -63 were abundantly
expressed in roots. In addition, the results also showed that most
of the maize CCCH genes in the subfamily I exhibited lower
expression levels in leaves and silks than in the other three tissues.
Since expression patterns can provide important clues for
possible gene function, we further investigated the expression
levels of these genes in response to abiotic stress by subjecting
three-week-old seedling leaves to ABA and drought treatments.
The results showed that expression levels of all seven genes were
induced or repressed by the two stress treatments, although the
induction of some genes was slight (Fig. 10A and B). Under ABA
treatment, ZmC3H4, -28, -43 and -54 were highly expressed at
a relatively early stage (3 h after treatment), whereas ZmC3H10
and -34 levels were peaked at 12 h after treatment. ZmC3H63 was
down-regulated by ABA treatment across all time points. Notably,
ZmC3H54 was strongly up-regulated (.2-fold) at 3 h after ABA
(Fig. 10A). Under drought stress, the highest expression levels of
ZmC3H28, -10, -34 and -54 were found at 3 h after treatment,
while those of ZmC3H4, -63 and -43 were observed later (12 h
after treatment). Similar to its response to ABA treatment,
ZmC3H54 was strongly up-regulated at 3 h after drought
treatment (Fig. 10B). In addition, ZmC3H43 was also significantly
up-regulated by drought stress. By comparing the expression
patterns of the two segmental duplicated genes (ZmC3H4 and -28)
in subfamily I, we found that the two duplicated genes exhibited
similar expression profiles following ABA treatment but showed
little difference under drought treatment.
CCCH-type zinc finger proteins comprise a large superfamily
and play important roles in many aspects of plant growth and
Figure 5. Phylogenetic relationships of maize, rice and
Arabidopsis CCCH proteins. The tree was constructed using the
ClustalX program by the N-J method from alignment of the full-length
amino acid sequences of maize, rice and Arabidopsis CCCH proteins.
Bootstrap values of 1,000 replications were executed, and only results
above 50% are shown at each node.
CCCH-Type Zinc Finger Family in Maize
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development. Compared to animals, few CCCH proteins have
been studied functionally in plants. In this study, we identified
68 CCCH genes in the maize genome and divided them into
seven groups based on phylogenetic analysis. This study of CCCH
proteins in maize confirms many patterns observed in other
species, but some novel features that differ from those seen in other
species were also found.
Previous studies revealed that CCCH proteins have between 1
and 6 CCCH zinc finger motifs defined as C-X6–14-C-X4–5-C-X3-
H . After analysis of the CCCH gene family in Arabidopsis and
Figure 6. Locations of 68 CCCH proteins on 10 maize chromosomes. The scale on the left is in megabases. Chromosome numbers are
indicated at the top of each bar. The gene names on the left side of each chromosome correspond to the approximate locations of each CCCH gene.
The chromosome order was arranged to bring duplicated regions in close proximity. The segmental duplication genes are connected by dashed lines,
and the tandem duplicated gene clusters are marked by red bars.
CCCH-Type Zinc Finger Family in Maize
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rice, these CCCH zinc finger motifs were redefined as C-X4–15-C-
X4–6-C-X3-H which are glycine-rich and phenylalanine-rich
sequences . As shown in this study, ZmC3H3 contains seven
copies of highly conserved CCCH motifs. Moreover, a novel C-
X17-C-X6-C-X3-H motif was identified in ZmC3H17. To our
knowledge, these striking features have not been reported in
Table 1. Ka/Ks analysis and estimated divergence time for the duplicated ZmC3H paralogs.
Duplicated pairs KaKs Ka/Ks Purifying selection Date (Mya)Duplicate type
ZmC3H14-ZmC3H460.044 0.143 0.308Yes 11Segmental
ZmC3H13-ZmC3H470.063 0.1680.375Yes 12.92 Segmental
ZmC3H16-ZmC3H570.04 0.2010.199Yes 15.46 Segmental
ZmC3H20-ZmC3H55 0.2050.2440.84 Yes18.77 Segmental
ZmC3H6-ZmC3H670.066 0.240.275 Yes18.46 Segmental
ZmC3H37-ZmC3H60 0.1340.131 1.023No 10.08Segmental
ZmC3H7-ZmC3H66 0.044 0.1750.251 Yes 13.46Segmental
ZmC3H29-ZmC3H360.014 0.169 0.083Yes13 Segmental
ZmC3H12-ZmC3H51 0.0590.172 0.343Yes 13.23 Segmental
ZmC3H39-ZmC3H53 0.061 0.178 0.343Yes 13.69Segmental
ZmC3H4-ZmC3H28 0.0250.23 0.109Yes 17.69Segmental
ZmC3H2-ZmC3H610.031 0.2050.151 Yes15.77 Segmental
ZmC3H18-ZmC3H56 0.095 0.0671.418 No5.15Segmental
ZmC3H23-ZmC3H640.067 0.3590.187 Yes27.62 Segmental
ZmC3H30-ZmC3H350.0530.156 0.34Yes 12Segmental
ZmC3H13-ZmC3H14 0.262 0.8180.32 Yes 62.92 Tandem
ZmC3H46-ZmC3H47 0.292 0.9650.303Yes 74.23 Tandem
Figure 7. Cis-elements in the promoter regions of stress-responsive CCCH genes. The stress-responsive cis-elements distributed on the
sense strand and reverse strand are shown above and below the black lines, respectively. ABRE and DRE core sequences are indicated by drop-down
arrows and triangles, respectively.
CCCH-Type Zinc Finger Family in Maize
PLoS ONE | www.plosone.org9July 2012 | Volume 7 | Issue 7 | e40120
previous studies. Generally speaking, these features are perhaps
exceptions, but they seem to characterize a new CCCH-type zinc
finger motif/protein in maize. To confirm this suggestion, further
biological experiments should be performed to examine their
biological functions in future studies. In maize, 79.4% of all
identified CCCH motifs are C-X7–8-C-X5-C-X3-H motifs, and
similar results were also found in the Arabidopsis and rice CCCH
gene families. Among the 68 maize CCCH proteins, 42 members
contain the C-X8-C-X5-C-X3-H motif and 37 members contain
the C-X7-C-X5-C-X3-H motif, suggesting the crucial functional
roles of these motifs in the CCCH proteins. A total of 180 CCCH
zinc finger motifs were identified in the maize CCCH family, more
than those of Arabidopsis (152) and rice (150) . Therefore, about
30 additional motifs were found in the maize CCCH family
compared to the Arabidopsis and rice CCCH families, despite the
three different plants having similar numbers of CCCH proteins.
To date, the origin of the 30 additional motifs in maize is
unknown, but we can speculate that they are perhaps a result of
plant adaptation to various regulatory processes during the
evolution of the genome. These novel observations provide
important references for exploring the evolutionary history and
functional mechanisms of CCCH proteins in plants.
Among the maize CCCH proteins phylogenetically analyzed in
this study, it was difficult to assign some genes into groups due to
their low bootstrap values in the clades (bootstrap values ,50%).
In fact, the unclear phylogenetic relationship with low bootstrap
support is similar to the phylogenetic analysis of CCCH proteins in
Arabidopsis and rice. Although sequence analysis revealed that the
CCCH motif is a highly conserved functional unit, their other
characteristics are highly diverse, especially in the different
numbers of CCCH motifs contained in each protein and the
spacing in the protein sequences between adjacent CCCH motifs
and adjacent cysteines of the zinc finger motif in each sequence.
Notably, the majority of CCCH proteins clustered in the same
groups displayed a similar domain architecture, suggesting their
similar subfamily-specific functions. A combined N-J tree was also
constructed to investigate the phylogenetic relationships of CCCH
proteins in the three representative species and their evolution-
function relationship. We noted that some ortholog proteins
displayed a closer relationship than paralog proteins among maize
and rice CCCH proteins, suggesting that the ortholog pairs may
have originated from a common ancestor when duplication events
occurred before the divergence of the grasses. Moreover, monocot
(maize and rice) and dicot (Arabidopsis) ortholog pairs were also
found in our phylogenetic analysis, indicating that these ortholog
pairs originated from common ancestral genes that existed before
the divergence of monocots and dicots. These observations
suggested that the ortholog genes are highly conserved during
Figure 8. Phylogenetic relationships and sequence analysis of stress-responsive CCCH genes. (A) The phylogenetic tree was constructed
using MEGA4.0 by the N-J method. Bootstrap values (above 50%) from 1,000 replicates are indicated at each node. (B) Sequence alignment of CCCH
zinc finger motifs of the stress-responsive CCCH genes. The conserved CCCH zinc finger motifs are indicated by straight lines. (C) Sequence alignment
of ANK repeat motifs of the stress-responsive CCCH genes. The conserved ANK motifs are indicated by straight lines.
CCCH-Type Zinc Finger Family in Maize
PLoS ONE | www.plosone.org10 July 2012 | Volume 7 | Issue 7 | e40120
the evolutionary process. Some groups of CCCH genes were
further expanded across the three species. We concluded that the
expanded CCCH genes may imply a correlation between gene
number expansion and their important roles in plant growth and
Wang et al. (2008) previously showed that segmental duplication
was largely responsible for the expansion of rice and Arabidopsis
CCCH gene families . In our study, a total of 15 sister gene
pairs of maize CCCH proteins were determined to be involved in
segmental duplications by shared phylogenetic clade combinations
within the same groups and by locations within the segmental
duplicated blocks . No obviously clustered CCCH genes were
detected on the 10 chromosomes besides the two sister pairs
ZmC4H13/14 and ZmC4H46/47. These results suggest that
segmental duplication is the main contributor to the expansion
of the maize CCCH family, which is consistent with the analysis of
the Arabidopsis and rice CCCH families . It is believed that
segmental duplication events occur more often in the more slowly
evolving gene families during the process of evolution . The
prevalence of ortholog pairs and segmental duplication in the
three different species suggested that the CCCH gene family is
a conserved and slowly evolving family in plant genomes. This
suggestion also gives us a possible reason or explanation for the
very similar number of CCCH proteins in the three species,
although the genome size of maize is approximately 18 and 6
times as that of Arabidopsis and rice, respectively [19,35]. Indeed,
gene duplication, including segmental and tandem duplications, is
one of the primary driving forces throughout the evolutionary
process of genomes . Previous studies showed that the maize
genome has undergone several rounds of whole genome duplica-
tion, including an ancient duplication prior to the divergence of
grasses (,50–70 Mya) and an additional whole genome duplica-
tion (,5 Mya), which distinguishes maize from sorghum [37,38].
By calculating the duplication dates of duplicated gene pairs, we
demonstrated that all of the segmental duplication events in the
maize CCCH family occurred after the divergence of the grasses.
Plant growth and productivity are frequently threatened by
environmental stresses such as drought, high salinity and low
temperature during their life cycles. Many stress-related genes are
induced in order to adapt to these environmental stresses [39,40].
In this study, seven maize CCCH genes were predicted to be
stress-related genes based on phylogenetic and sequence analysis,
and their expression patterns were investigated in five represen-
tative tissues and under ABA and drought treatments. The results
showed that the seven maize CCCH genes were found to be
broadly expressed in almost all tissues examined. Although the
seven genes have close evolutionary relationships, they showed
largely differential expression patterns in the five tissues. This
finding suggested that these genes may function at different stages
of plant growth and development. In addition, all seven genes
were found to be responsive to ABA treatment or drought stress,
and ZmC3H54 was strongly inducted by both stimuli. We
concluded that ZmC3H54 may play an essential role in response
to abiotic stresses. This conclusion was supported by the close
relationship of ZmC3H54 to ATSZF1, ATSZF2 and CaKR1,
which were previously identified as stress-response genes [16,29].
Figure 9. Expression of seven stress-responsive CCCH genes in five representative maize tissues. The X-axis is the representative tissues:
R, root; E, ear; L, leaf; S, stem; and SK, silk. The Y-axis is the scale of relative expression level of the gene compared to the expression level in leaf. Error
bars, 6 SE.
CCCH-Type Zinc Finger Family in Maize
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We noted that the two segmental duplication genes, ZmC3H4 and
-28, exhibited similarly expression levels in response to ABA
treatment. Although the peak expression levels of ZmC3H4 and -
28 were found at different time points, they were largely up-
regulated under drought treatment. Meanwhile, the Ka/Ks ratio
of this segmental pair was only 0.109. Therefore, we concluded
Figure 10. Expression of seven stress-responsive CCCH genes under stress treatments. The Y-axis is the scale of relative expression levels.
The X-axis is time courses of stress treatments. Error bars, 6 SE. (A) Realtive expression levels of the seven stress-responsive CCCH genes in response
to ABA treatment. Seedlings were sampled at 0 h (CK), 3 h (A1), 6 h (A2) and 12 h (A3) after ABA treatment. (B) Realtive expression levels of the seven
stress-responsive CCCH genes in response to drought stress. Seedlings were sampled at 0 h (CK), 3 h (D1), 6 h (D2) and 12 h (D3) after drought
CCCH-Type Zinc Finger Family in Maize
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that ZmC3H4 and -28 may exert redundant functions in response
to abiotic stress. We also noted that some genes were not only
induced by the two stress treatments, but they were also
abundantly expressed in specific tissues. These results suggested
that these genes may be important both for stress responses and
developmental processes. Intriguingly, the results of qRT-PCR
were not always consistent with those of the promoter sequence
analysis. For example, the promoter analysis showed that ZmC3H4
and -34 contain one type of stress-responsive cis-element in their
promoter regions (Fig. 7), but these genes were induced by both
ABA and drought treatments. Thus, the possible regulatory
mechanisms of ZmC3H4 and -34 in responses to stresses remain
unclear. We concluded that some unidentified stress-responsive cis-
elements may be present in the promoters of these two genes.
Alternatively, ZmC3H4 and -34 may be secondary targets, which
are indirectly regulated by ABA or drought. We will experimen-
tally examine their biological functions in future studies.
In conclusion, CCCH-type zinc finger proteins have essential
functions in various developmental processes in plants. To date,
the regulatory mechanisms of CCCH proteins in plants remain
poorly understood. Therefore, the systematic analysis of the
CCCH gene family provides an important reference for future
studies on the biological functions of maize CCCH proteins.
Materials and Methods
Identification and Sequence Analysis of CCCH Proteins in
Our identification of non-redundant maize CCCH zinc finger
proteins was performed using the following strategy. Maize
genome sequenceswere downloaded
maizesequence.org/index.html, and then DNATOOLS software
was used to construct a local database from the nucleotide
sequences and protein sequences of the latest complete maize
genome. Sequences of Arabidopsis and rice CCCH proteins
representing different CCCH motif types of C-X4–15-C-X4–6-C-
X3-H were used as queries to search against the maize protein
database with BLASTP program . All hits with E-values below
0.001 were selected for further analysis, and this step was crucial to
identifying all possible maize CCCH proteins. All candidate
sequences that met the standards were confirmed to be real
CCCH proteins by Pfam (PF00642) (http://pfam.sanger.ac.uk/)
andSMART (Sm00356) (http://smart.embl-heidelberg.de/)
[41,42]. Finally, all of the confirmed CCCH proteins were aligned
using ClustalW , and all identical sequences were checked
manually to remove redundant sequences.
Information on maize CCCH genes, including BAC number,
chromosomal location, coding sequence (CDS), exons and introns
number, ORF length and amino acid (AA), was obtained from the
maize B73 sequencing database. The molecular weight (kDa) and
isoelectric point (PI) of each gene were calculated by online
ExPASy programs (http://www.expasy.org/tools/). Sequences
logos of the CCCH zinc finger motifs were produced by online
WebLogo software . Exon-intron structure analysis was
deduced using GSDS (http://gsds.cbi.pku.edu.cn/) . Maize
CCCH genes were placed on chromosomes according to their
starting positions given in the maize B73 sequencing database.
Chromosome location image was generated by MapInspect
html). To predict cis-acting regulatory DNA elements (cis-elements)
in promoter regions of CCCH genes, the PLACE website (http://
www.dna.affrc.go.jp/PLACE/signalscan.html) was adopted to
identify putative cis-elements in the 2,000 bp genomic DNA
sequences upstream of the initiation codon (ATG) .
Full-length sequences of maize, rice and Arabidopsis CCCH
proteins were aligned using ClustalX v1.83 . Phylogenetic
trees were conducted using MEGA 4.0 . The neighbor-joining
(N-J) method was used to construct different trees with the pairwise
deletion option. For statistical reliability, bootstrap analysis was
carried out with 1,000 replicates to evaluate the significance of
each node. For detection of tandem and segmental duplications,
paralogs were regarded as tandem duplicated genes provided two
maize CCCH genes were separated by five or fewer gene loci
according to the maize B73 genome annotation. Paralogs were
designated as segmental duplicated genes if they were placed on
duplicated chromosomal blocks as previously proposed by Wei et
al. (2007) [22,49,50,51].
The number of nonsynonymous substitutions per nonsynon-
ymous site (Ka) and the number of synonymous substitution per
synonymous site of duplicated genes were calculated by DnaSP
v5.0 . The ratio of nonsynonymous to synonymous nucleotide
substitutions (Ka/Ks) between paralogs was analyzed to detect the
mode of selection. To estimate the times of duplication events, the
Ks value was translated into duplication time in million years
based on a rate of l substitutions per synonymous site per year.
The duplication time (T) was calculated as T
2l61026Mya (l=6.561029for grasses) [23,24].
Plant Materials, Growth Condition and Stress Treatments
The maize inbred line B73 was used to check the gene
expression levels in all experiments. Five representative tissues (10-
day-old root, 3-week-old leaf, 6-week-old stem, 5–8-cm young ear
and 6–10-cm silk) were collected from a life cycle of maize. For
expression analysis of maize CCCH genes under stress, plants
were grown in a greenhouse with a 14-h light/10-h dark cycle at
28–30uC. The drought treatment was performed following the
method described in our previous study . For ABA treatment,
3-week-old seeding leaves were sprayed with 100 mM ABA
solution and sampled at 0, 3, 6 and 12 h after treatment
[53,54]. For all the stages, three biological replicates were
conducted for each sample.
RNA Extraction and qRT-PCR Analysis
Total RNAs of all the collected samples were extracted using the
Trizol reagent (Invitrogen) according to the manufacturer’s
instructions. The DNase-treated RNA was reverse-transcribed
using M-MLV reverse transcriptase (Invitrogen). qRT-PCR was
performed on an ABI 7300 Real-Time system (Applied Biosys-
tems). The gene-specific primers designed using Primer Express
3.0 software (Applied Biosystems) were employed to amplify 90–
150 bp PCR products unique to each gene (Table S3). Each
reaction contained 12.5 ml of 26SYBR Green Master Mix reagent
(Applied Biosystems), 2.0 ml of diluted cDNA sample, and 400 nM
gene-specific primers in a final volume of 25 ml. The thermal cycle
was used as follows: 95uC for 10 min, followed by 40 cycles at
95uC for 15 s and 60uC for 1 min. After the PCR was completed,
a melting curve was generated to analyze the specificity for each
gene by increasing the temperatures from 60 to 95uC. Three
technical replicates were performed for each gene. Expression
level of the maize Actin 1 gene was used as an internal control. The
relative expression level was calculated as 2–DDCT[DCT = CT,
Target– CT, Actin 1. DDCT = DCT, treatment– DCT, CK (0h)]. The
relative expression level (2–DDCT, CK (0h)) in the normal plant
without stress treatment was normalized to 1 as described
previously . Statistical analyses were performed using SDS
software 1.3.1 (Applied Biosystems).
CCCH-Type Zinc Finger Family in Maize
PLoS ONE | www.plosone.org13 July 2012 | Volume 7 | Issue 7 | e40120
CCCH gene family in maize.
Detailed information of CCCH proteins in
Maize CCCH gene-specific primers used for
We thank members of the Key Laboratory of Crop Biology of Anhui
province for their assistance in this study.
Conceived and designed the experiments: XJP YZ BJC. Performed the
experiments: XJP YZ JGC. Analyzed the data: XJP YZ WZ HYJ XYL.
Wrote the paper: XJP YZ BJC. Participated in the design of this study and
revised the manuscript: QM SWZ.
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