Mol Genet Genomics (2014) 289:855–872
DOI 10.1007/s00438-014-0858-9
ORIGINAL PAPER
Comprehensive analysis of CCCH‑type zinc finger gene family
in citrus (Clementine mandarin) by genome‑wide characterization
Shengrui Liu · Muhammad Rehman Gul Khan ·
Yongping Li · Jinzhi Zhang · Chungen Hu 
Received: 8 January 2014 / Accepted: 19 April 2014 / Published online: 13 May 2014
© Springer-Verlag Berlin Heidelberg 2014
Abstract  The CCCH-type zinc finger proteins comprise
a large gene family of regulatory proteins and are widely
distributed in eukaryotic organisms. The CCCH proteins
have been implicated in multiple biological processes and
environmental responses in plants. Little information is
available, however, about CCCH genes in plants, espe-
cially in woody plants such as citrus. The release of the
whole-genome sequence of citrus allowed us to perform a
genome-wide analysis of CCCH genes and to compare the
identified proteins with their orthologs in model plants. In
this study, 62 CCCH genes and a total of 132 CCCH motifs
were identified, and a comprehensive analysis including the
chromosomal locations, phylogenetic relationships, func-
tional annotations, gene structures and conserved motifs
was performed. Distribution mapping revealed that 54 of
the 62 CCCH genes are unevenly dispersed on the nine
citrus chromosomes. Based on phylogenetic analysis and
gene structural features, we constructed 5 subfamilies of
62 CCCH members and integrative subfamilies from cit-
rus, Arabidopsis, and rice, respectively. Importantly, large
numbers of SNPs and InDels in 26 CCCH genes were
identified from Poncirus trifoliata and Fortunella japonica
using whole-genome deep re-sequencing. Furthermore,
citrus CCCH genes showed distinct temporal and spatial
Communicated by S. Hohmann.
Electronic supplementary material  The online version of this
article (doi:10.1007/s00438-014-0858-9) contains supplementary
material, which is available to authorized users.
S. Liu · M. R. G. Khan · Y. Li · J. Zhang · C. Hu (*)  gene families. So far, only the MADS-Box family, the
Key Laboratory of Horticultural Plant Biology (Ministry
of Education), College of Horticulture and Forestry Science,
Huazhong Agricultural University, Wuhan 430070, China
e-mail: chungen@mail.hzau.edu.cn
expression patterns in different developmental processes
and in response to various stress conditions. Our compre-
hensive analysis of CleC3Hs is a valuable resource that fur-
ther elucidates the roles of CCCH family members in plant
growth and development. In addition, variants and com-
parative genomics analyses deepen our understanding of
the evolution of the CCCH gene family and will contribute
to further genetics and genomics studies of citrus and other
plant species.
Keywords  CCCH zinc finger · Phylogenetic analysis ·
Gene structure and conserved motifs · SNPs and InDels ·
Expression patterns · Clementine mandarin
Introduction
Citrus trees are the most widely grown and economically
important fruit crop in the world. Most citrus species are
diploid (2n = 2x = 18) with highly heterozygous, relatively
small genomes and over 30,000 predicted genes (Gmitter
et al. 2012). All citrus species belong to the Rutaceae fam-
ily, which includes six closely related genera: Citrus, For-
tunella, Poncirus, Eremocitrus, Microcitrus and Clymenia
(Swingle and Reece 1967). Despite the economic impor-
tance of citrus, genome-wide research resources and studies
have been limited. However, the recent availability of the
complete genome sequences and assemblies of clementine
mandarin (Gmitter et al. 2012) and sweet orange (Xu et al.
2013) now allow for comprehensive bioinformatics analy-
ses and the characterization of numerous known or novel
members of which are involved in plant development and
signal transduction, has been comprehensively analyzed
(Hou et al. 2014). The CCCH zinc finger proteins associate
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with RNA and are important regulators involved in plant
development and stress responses, but there have been no
previous studies to functionally characterize CCCH genes
in citrus. Therefore, we took advantage of recent genomic
advancements to analyze the CCCH genes in the whole cit-
rus genome.
As one of the largest transcription factor families in
plants, the zinc finger transcription factors are important
regulators involved in plant development and biological
processes. They are characterized by the presence of com-
mon zinc finger motif, which play critical roles in inter-
actions with other molecules (Takatsuji 1998; Moore and
Ullman 2003). Most zinc finger transcription factors pre-
viously identified in plants, such as the RING-finger, Dof,
WRKY, ERF, and LIM families, regulate gene expres-
sion by binding DNA or proteins (Kosarev et al. 2002;
Lijavetzky et al. 2003; Zhang and Wang 2005; Nakano
et al. 2006; Arnaud et al. 2007). In contrast, CCCH zinc
fingers are specific and distinct from other zinc finger tran-
scription factors, which regulate gene expression by bind-
ing to mRNA (Wang et al. 2008a).
The CCCH domain proteins contain 1–6 typical C3H-
type motifs and were originally defined as C–X6–14–C–
X4–5–C–X3–H (Berg and Shi 1996), but redefined as
C–X4–17–C–X4–6–C–X3–H recently (Peng et al. 2012).
The well-studied mammalian protein tristetraprolin, a
member of the TIS11 family, contains two CCCH zinc fin-
gers and can directly bind to AU-rich elements within the
3′-untranslated region of the target transcripts to facilitate
mRNA degradation (Lai et al. 1999, 2000, 2003; Baou
et al. 2009). The identified Zfp36l2 protein in mouse, which
is an mRNA-binding and destabilizing protein, plays a vital
role in the physiological control of female fertility at the
level of early embryonic development (Ramos et al. 2004;
Stumpo et al. 2009). In Caenorhabditis elegans, PIE-1 and
POS-1 are characterized as two CCCH proteins that can
control germ cell fate by inhibiting transcription or the acti-
vation of protein expression from maternal RNAs (Tenen-
haus et al. 2001; Ogura et al. 2003). Another CCCH pro-
tein, zinc finger antiviral protein, has been isolated from
Rat2 fibroblasts, can directly bind to specific viral RNA
sequences through its CCCH motifs and inhibit retroviral
RNA production (Gao et al. 2002).
Although many CCCH proteins have been characterized
in animals, only a small number have been functionally
characterized in plants. CCCH proteins have a large degree
of functional diversity and are involved in a wide range
of biological processes, such as embryo development (Li
and Thomas 1998), floral morphogenesis (Li et al. 2001),
FRIGIDA-mediated winter-annual habit (Schmitz et al.
2005), salt stress responses (Sun et al. 2007), seed germina-
tion (Kim et al. 2008), and secondary cell wall biosynthesis
(Ko et al. 2009) in Arabidopsis. In rice, many CCCH genes
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Mol Genet Genomics (2014) 289:855–872
are involved in processes such as leaf senescence (Kong
et al. 2006) and plant architecture determination (Wang
et al. 2008b). In particular, OsC3H12 (Os01g68860) has
shown that enhanced resistance to bacterial blight disease
accompanied by the accumulation of jasmonic acid (JA)
and induced expression of JA signaling genes (Deng et al.
2012), as well as OsTZF1 confers delayed senescence and
stress tolerance by regulating stress-related genes (Jan et al.
2013). Besides, CsSEF1 was characterized, which encod-
ing putative CCCH-type zinc finger protein expressed dur-
ing cucumber somatic embryogenesis (Grabowska et al.
2009). Another CCCH protein from cotton, GhZFP1, inter-
acts with GZIRD21A and GZIPR5 to enhance tolerance to
drought, salt, salicylic acid, and fungal disease stresses in
transgenic plants (Guo et al. 2009).
The current study was endeavor toward genomic identi-
fication of all CCCH domain-containing genes in citrus to
enhance our understanding of their functions. We identified
62 CCCH genes by studying sequence phylogeny, genome
organization, chromosomal location, gene structure, and
conserved motifs. We also identified SNPs and InDels in
CCCH genes between different citrus species. In addition,
24 genes were selected for investigation of their expression
patterns in different tissues. The expression patterns of sev-
eral genes were further surveyed under drought and ABA
stress conditions. Our results provide a subset of candidate
genes that may be used for future investigation into the
functions of CCCH genes in citrus development and stress
response.
Methods
Identification of CCCH genes in citrus
Citrus clementine genome database (http://www.phytozome.
net/clementine.php) was employed to identify CCCH
motif-containing proteins using the Basic Local Alignment
Search Tool algorithms (BLASTP) and TBLASTN with the
published Arabidopsis, rice, Populus, human and Trypano-
soma CCCH proteins as query sequence and with e-value
cutoff set as 1e−005 (Hudson et al. 2004; Wang et al.
2008a; Kramer et al. 2010). The Hidden Markov Model
of Simple Modular Architecture Research Tool (SMART)
(Letunic et al. 2004) and Pfam (Finn et al. 2006) were used
to examine all obtained protein sequences for the pres-
ence of CCCH motif. The sequences identified by SMART
(Sm00356) and Pfam (PF00642) were considered to be cit-
rus CCCH proteins. Online web server FGENESH (Sala-
mov and Solovyev 2000) was used to correct the predicted
genes by manual re-annotation. The sequences were further
examined for the CCCH domain using the InterProScan
program (Quevillon et al. 2005) with stringent parameters.
Mol Genet Genomics (2014) 289:855–872 857
We identified 62 unique CCCH motif-containing genes
and named them CleC3H01 to CleC3H62, following the
nomenclature proposed by Hu et al. (2010). The number of
CCCH genes we identified is far more than that predicted
in PlantTFDB (Zhang et al. 2011a). To further character-
ize the citrus CCCH domain-containing proteins, an online
ExPasy program (http://web.expasy.org/protparam/) was
used to calculate the length, molecular weight, and isoelec-
tric point of each protein.
Phylogenetic analysis of citrus CCCH genes
The ClustalX (version2.1) program was used to generate
multiple alignments of the 62 amino acid sequences. Errors
were manually corrected. The phylogenetic trees were gen-
erated with MEGA4.0 using the Neighbor-Joining (NJ)
algorithm (Tamura et al. 2007). Bootstrap analysis with
1,000 replicates was used to evaluate the significance of the
nodes. Pairwise gap deletion mode was used to ensure that
the divergent domains could contribute to the topology of
the NJ tree.
Functional assignments and sequence properties of citrus
CCCH genes
To assign putative functions to the CCCH genes, the
Blast2go program was run locally to BLAST against a ref-
erence database that stores UniProt entries, Gene Ontology,
Enzyme Commission, and Kyoto Encyclopedia of Genes
and Genomes annotation (Schmid and Blaxter 2008). The
amino acid sequences of CCCH proteins were analyzed for
physicochemical parameters (ProtParam). The exon/intron
organization of CCCH genes were identified by compar-
ing the coding sequences with their corresponding genomic
sequences using Gene Structure Display Server program
(Guo et al. 2007). SMART (Letunic et al. 2004) and Mul-
tiple EM for Motif Elicitation were used to identify con-
served motif structures of CCCH protein sequences.
Chromosomal location of citrus CCCH genes
To determine the physical location of CCCH genes, the
starting position of all CCCH genes on each chromosome
was confirmed by BlastN searches against the local data-
base of the complete sequence of the sweet orange genome
(Xu et al. 2013). MapInspect software (http://www.plantb
reeding.wur.nl/uk/softwaremapinspect.html) was used to
confirm chromosomal location of citrus CCCH genes and
results were revised manually. The duplication events of
CCCH genes in citrus were searched based on previous
parameters: e-value <1e−10 and identity >90 % (Song
et al. 2013).
Plant material and treatments
Clementine mandarin was grown in the greenhouse (16 h
light/8 h dark, 25 ± 2 °C) in a 3:1 soil: sand mixture. Seed-
lings that germinated after 6 weeks were used for sample
collection from different tissues and for different stress
treatments. For drought stress, the seedlings were treated
with 300 mM mannitol and samples were collected at 0, 1,
6, 12, 24 and 48 h after treatment. For ABA treatment, the
seedling leaves were sprayed with 100 μM ABA and sam-
ples were collected at 0, 0.5, 1, 2, 4 and 6 h after treatment.
Treatment with deionized water was performed as a control
at 0 h point. The control and stress-treated plants were col-
lected, frozen in liquid nitrogen, and stored at −80 °C to
await further analysis. At least three biological replicates
were performed for each sample at all developmental stages.
Real‑time PCR verification
Total RNA was isolated using Oligotex mRNA mini kit
(Qiagen, USA) according to the manufacturer’s instruc-
tions. Total RNA was treated with DNase I and first strand
synthesis of cDNA was performed using RT Primer Mix
and Primescript RT Enzyme Mix I. The gene-specific
primers were designed using Primer 5.0 with melting tem-
peratures of 58–60 °C, primer lengths of 19–20 bp and
amplicon lengths with 91–242 bp. Real-time RT-PCR was
conducted on LightCyclerW480 Detection System (Roche,
Germany), as described previously (Zhang et al. 2011b).
At least three replicates were performed for each gene. The
expression level of the citrus β-actin was used as the inter-
nal reference gene. Relative gene expression with respect
to β-actin was determined as described previously (Livak
and Schmittgen 2001). One-way ANOVA was performed
by SPSS to obtain the P values.
Results
Characterization and analysis of citrus CCCH genes
All the proteins containing the motif C–X4–15–C–X4–6–C–
X3–H were selected in citrus genome database. This motif
covers both the conventional (C–X7–C–X5–C–X3–H and
C–X8–C–X5–C–X3–H) and the recently defined non-con-
ventional CCCH motifs. The HMM profiles of the CCCH
genes from Arabidopsis, rice, Populus, human, and Trypa-
nosoma were used as query to identify the CCCH motif-
containing genes using BLASTP and TBLASTN pro-
grams. A total of 62 non-redundant CCCH genes (named
CleC3H01 to CleC3H62) were obtained. The number
of identified citrus CCCH genes (62) differs from other
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representative species, such as Populus, Arabidopsis, rice,
maize, mouse, human and Trypanosoma brucei, which
contain 91, 68, 67, 68, 58, 55 and 48 previously predicted
CCCH genes, respectively (Hudson et al. 2004; Liang et al.
2008; Wang et al. 2008a; Kramer et al. 2010; Chai et al.
2012; Peng et al. 2012). The 62 identified citrus CCCH
genes are presented in Table 1, which includes the length
of the Open Reading Frame (ORF), the number of exons,
molecular weights, isoelectric points (PIs), and the CCCH
motif number of each protein. These CCCH genes are pro-
tein coding and the length of amino acid (aa) sequences
ranges from 121 (CleC3H25) to 2,165 (CleC3H07) with an
average of 556 aa. The isoelectric points varied from 4.75
(CleC3H20) to 9.67 (CleC3H61). Further details on the
sequences and motifs of the 62 CCCH genes are provided
in Online Source 1.
Previous studies indicate that members of the CCCH
gene families in both animals and plants have between
one and six CCCH motifs (Hudson et al. 2004; Wang et al.
2008a; Kramer et al. 2010; Chai et al. 2012). In this study,
we identified the motif characteristics of CCCH genes in
citrus, Arabidopsis, rice and Populus (Fig. 1). Similar to the
other three species (Arabidopsis, rice and Populus), all of
the citrus CCCH genes have between one and six CCCH
motifs and 58.1 % of them have at least two CCCH motifs.
A total of 132 CCCH motifs were identified (Table 2),
which were fewer than those found in Arabidopsis (152),
rice (150), Populus (211) and maize (180). Two conven-
tional CCCH motifs (C–X7–C–X5–C–X3–H and C–X8–C–
X5–C–X3–H) accounted for 82.3 % of all the citrus CCCH
motifs and constituted the largest two groups, similar to
those found in Arabidopsis (82.2 %), rice (78.7 %), Pop-
ulus (82.0 %) and maize (79.4 %), followed by C–X5–C–
X4–C–X3–H and C–X7–C–X4–C–X3–H. It is noteworthy
that the C–X7–C–X6–C–X3H motif only has been identified
in Arabidopsis, rice and maize as compared with in citrus
and Populus. Surprisingly, we did not find unique motifs
in citrus as compared with Arabidopsis, while the C–X10–
C–X5–C–X3H motif is unique to citrus as compared with
Populus.
Chromosomal location and gene duplication of citrus
CCCH genes
Previous studies have shown that the clementine manda-
rin is a hybrid of the ‘Mediterranean’ mandarin (C. reticu-
lata)  × sweet orange (tangor) (Nicolosi et al. 2000; Olli-
trault et al. 2012; Xu et al. 2013). Therefore, the CCCH
genes were located in the sweet orange genome. According
to the starting position of each gene, 54 of the 62 CCCH
genes were found to be unevenly distributed across the 9
citrus chromosomes, whereas the chromosomal location of
the 8 remaining genes (CleC3H04, 12, 28, 35, 36, 37, 56
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Mol Genet Genomics (2014) 289:855–872
and 58) is unknown (Fig. 2). Among these 54 genes, chro-
mosome 2 contains the largest number of CCCH genes
(12). By contrast, chromosomes 1, 3, 6, and 9 each have
fewer CCCH genes; 2, 4, 5 and 3, respectively. Chromo-
somes 4 and 7 each contain 8 CCCH genes while chromo-
somes 5 and 8 both have 6 (Fig. 2).
Inspection of the phylogenetic tree topology uncov-
ered several pairs of CCCH proteins with a high degree
of homology, suggesting that they are putative paralogous
pairs (homologous genes within a species that diverged by
gene duplication) (Fig. 3). The identification of paralogs
was based on the following criteria: (1) the length of aligned
sequence covers >80 % of the longer gene; and (2) the simi-
larity of the aligned regions is >70 % (Gu et al. 2002; Yang
et al. 2008). In total, only six pairs (CleC3H28/CleC3H29,
CleC3H35/CleC3H36, CleC3H08/CleC3H56, CleC3H46/
CleC3H58, CleC3H21/CleC3H22 and CleC3H57/CleC3H60)
of putative paralogous CCCH proteins were identified,
accounting for <20 % of the entire family, with sequence
similarity of 76.4, 77.8, 79.7, 83.6, 91.1, 96.6 %, respec-
tively. Further alignment analysis revealed that only the
CleC3H57/CleC3H60 gene pair exhibited a higher sequence
similarity (amino acid identity >90 %) based on strin-
gent criteria. The distance between the two genes is <4 kb
on chromosome 1, which provides evidence for a tandem
duplication.
Phylogenetic analysis of the citrus CCCH genes
To evaluate evolutionary relationships among the CCCH
genes, a phylogenetic analysis was performed based on the
full-length amino acid sequences from citrus, Arabidop-
sis and rice. Perhaps, owing to the divergence of CCCH
domains (e.g., the diverse CCCH motif types that possess
different spacing amino acids between conserved Cys and
His residues in each protein) and other non-homologous
motifs (e.g., ANK, RRM, WD), the obtained resultant tree
had low sequence similarity overall. The genes were mainly
divided into nine subfamilies and some of members did
not show significant similar gene structure and conserved
motifs, which does not support real evolutionary relation-
ships between different subfamilies (Online Source 2).
To gain further insight into the evolutionary relationships
between these subfamilies, all the CCCH genes of the three
species were divided into five subfamilies and named as
CCCH-a, b, c, d and e according to a previously described
method (Chai et al. 2012). The five subfamilies CCCH-
a (1–3 C–X7–C–X5–C–X3–H), CCCH-b (1–6 C–X8–C–
X5–C–X3–H), CCCH-c (2–3 C–X7–C–X5–C–X3–H and
C–X8–C–X5–C–X3–H), CCCH-d (1 C–X5–C–X4–C–X3–H
and 1 C–X7,8,10–C–X5–C–X3–H) and CCCH-e (1–6 other
non-conventional CCCH motifs) contained 47, 59, 25, 28
and 37 members, respectively (AtC3H13 was excluded due
Mol Genet Genomics (2014) 289:855–872 859

Table 1  List of 62 CCCH genes identified in citrus and their characteristics

Gene name Locus name Arabidopsis orthologs ORF (bp) Exons Protein Number of CCCH Subfamily
locus motif
Length (aa) Wt (kD) PI

CleC3H01 Ciclev10015314m.g AT1G03790.1 1,290 1 429 48.25 7.00 2 d

CleC3H02 Ciclev10015215m.g AT1G04990.1 1,356 7 451 48.78 8.25 5 b
CleC3H03 Ciclev10028282m.g AT1G07360.1 1,485 4 494 55.82 7.91 1 a
CleC3H04 Ciclev10003415m.g AT1G10320.1 2,265 11 755 88.90 7.62 2 e
CleC3H05 Ciclev10010745m.g AT1G10320.1 1,020 7 339 39.43 6.71 2 e
CleC3H06 Ciclev10000970m.g AT1G19860.1 1,380 4 459 52.99 6.31 1 b
CleC3H07 Ciclev10000009m.g AT1G21570.1 6,498 10 2,165 237.90 8.68 5 e
CleC3H08 Ciclev10012216m.g AT1G27650.1 963 3 320 37.14 9.65 2 c
CleC3H09 Ciclev10029043m.g AT1G27650.1 819 5 272 32.24 9.42 1 a
CleC3H10 Ciclev10014454m.g AT1G30460.1 2,106 7 701 76.34 6.23 3 e
CleC3H11 Ciclev10015789m.g AT1G32360.1 1,050 2 349 38.49 7.16 3 c
CleC3H12 Ciclev10003479m.g AT1G66810.1 834 3 277 30.2 9.07 1 b
CleC3H13 Ciclev10013897m.g AT1G66810.1 906 2 301 34.63 6.33 2 b
CleC3H14 Ciclev10015908m.g AT1G68200.1 984 2 327 36.49 7.66 2 b
CleC3H15 Ciclev10008647m.g AT1G75340.1 1,143 10 380 41.1 8.52 1 a
CleC3H16 Ciclev10004417m.g AT2G02160.1 2,214 3 737 81.82 5.51 3 e
CleC3H17 Ciclev10007946m.g AT2G02160.1 1,620 3 539 60.77 7.94 3 e
CleC3H18 Ciclev10014902m.g AT2G05160.1 1,575 8 524 59.53 5.96 1 a
CleC3H19 Ciclev10007876m.g AT2G05160.1 1,698 7 565 63.84 6.16 1 a
CleC3H20 Ciclev10027673m.g AT2G16470.1 5,337 10 1,778 192.81 4.75 1 a
CleC3H21 Ciclev10031903m.g AT2G20280.1 1,092 8 363 41.46 5.16 1 a
CleC3H22 Ciclev10012046m.g AT2G20280.1 1,080 8 359 41.17 5.75 1 a
CleC3H23 Ciclev10008089m.g AT2G24830.1 1,494 4 497 56.65 5.61 1 a
CleC3H24 Ciclev10027798m.g AT2G28450.1 2,583 14 860 93.64 5.33 1 b
CleC3H25 Ciclev10003110m.g AT2G32930.1 366 2 121 14.18 5.32 1 b
CleC3H26 Ciclev10014553m.g AT2G33835.1 1,950 5 649 71.67 8.24 1 a
CleC3H27 Ciclev10030920m.g AT2G40140.1 1,965 1 654 71.57 6.74 2 d
CleC3H28 Ciclev10004423m.g AT2G41900.1 2,196 1 731 79.27 6.09 2 d
CleC3H29 Ciclev10030816m.g AT2G41900.1 2,178 1 725 79.00 6.12 2 d
CleC3H30 Ciclev10018660m.g AT2G47680.1 3,063 14 1,020 114.44 6.15 2 c
CleC3H31 Ciclev10019968m.g AT2G47850.1 1,434 7 477 51.09 9.10 5 b
CleC3H32 Ciclev10012993m.g AT2G47850.2 468 5 155 17.42 8.8 1 b
CleC3H33 Ciclev10011741m.g AT3G02830.1 1,320 7 439 47.86 8.80 5 b
CleC3H34 Ciclev10031666m.g AT3G08505.1 1,248 5 415 47.30 6.57 4 e
CleC3H35 Ciclev10012360m.g AT3G12130.1 873 3 290 30.57 9.52 3 c
CleC3H36 Ciclev10016092m.g AT3G12130.1 903 3 300 31.73 9.37 3 c
CleC3H37 Ciclev10004790m.g AT3G12680.1 1,344 11 447 55.06 6.97 6 b
CleC3H38 Ciclev10018493m.g AT3G18640.1 4,320 6 1,439 158.05 8.71 1 a
CleC3H39 Ciclev10008851m.g AT3G19360.1 1,017 2 338 37.82 7.50 3 c
CleC3H40 Ciclev10014173m.g AT3G27700.1 2,841 4 946 103.52 6.42 1 b
CleC3H41 Ciclev10028940m.g AT3G47120.1 894 4 297 34.24 9.08 1 a
CleC3H42 Ciclev10019979m.g AT3G48440.1 1,431 6 476 53.16 5.12 5 b
CleC3H43 Ciclev10030728m.g AT3G51950.1 2,424 8 807 89.72 8.77 1 a
CleC3H44 Ciclev10018237m.g AT3G51950.1 1,920 8 639 70.82 6.20 1 a
CleC3H45 Ciclev10005002m.g AT4G25440.1 1,305 9 434 47.11 8.08 2 e
CleC3H46 Ciclev10024419m.g AT4G25440.1 1,311 7 436 47.49 7.63 2 e
CleC3H47 Ciclev10017981m.g AT4G25440.1 1,035 6 344 38.60 8.43 1 a

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Table 1  continued
Gene name Locus name Arabidopsis orthologs ORF (bp) Exons Protein Number of CCCH Subfamily
locus motif
Length (aa) Wt (kD) PI
CleC3H48 Ciclev10008633m.g AT4G29190.1 1,146 1 381 42.41 6.32 2 d
CleC3H49 Ciclev10027934m.g AT4G38890.1 2,082 10 693 76.99 6.93 1 e
CleC3H50 Ciclev10021165m.g AT5G06420.1 972 3 323 36.59 6.84 1 b
CleC3H51 Ciclev10006979m.g AT5G07500.1 789 3 262 30.24 9.28 2 d
CleC3H52 Ciclev10014515m.g AT5G12440.1 2,001 7 666 73.73 5.81 1 a
CleC3H53 Ciclev10014437m.g AT5G12850.1 2,139 1 712 78.15 6.29 2 d
CleC3H54 Ciclev10016232m.g AT5G16540.1 825 6 274 30.53 8.60 4 b
CleC3H55 Ciclev10004882m.g AT5G18550.1 1,431 7 476 50.11 8.63 5 b
CleC3H56 Ciclev10003384m.g AT5G42820.2 975 1 324 38.33 9.60 2 c
CleC3H57 Ciclev10025568m.g AT5G49200.1 1,380 9 459 52.26 9.15 1 a
CleC3H58 Ciclev10004933m.g AT5G51980.1 1,389 7 462 51.06 6.24 2 e
CleC3H59 Ciclev10010506m.g AT5G56900.2 1,914 10 637 70.68 6.33 2 b
CleC3H60 Ciclev10007413m.g AT5G56930.1 2,640 7 879 96.88 6.11 3 c
CleC3H61 Ciclev10027460m.g AT5G56930.1 1,164 7 387 44.33 9.67 1 a
CleC3H62 Ciclev10027883m.g AT5G58620.1 2,226 1 741 81.06 5.99 2 d

bp base pair, aa amino acids, D Dalton

Fig. 1  Numbers of CCCH
proteins with 1, 2, 3, 4, 5 or 6
CCCH motifs from Clementine
(Cle), Arabidopsis (At), Rice
(Os) and Populus (Pt)

to low similarity) (Fig. 4). The phylogenetic tree was con-
structed based on the full-length protein sequences using
Neighbor-Joining (NJ) for each subfamily. In addition,
Minimal Evolution (ME) and Maximum Parsimony (MP)
algorithms were also used to construct each subfamily,
but the tree topologies constructed by the three algorithms
were almost identical, save for the interior branches (data
not shown). Therefore, only the NJ phylogenetic tree was
used for further investigation in our study.
The phylogenetic trees indicate that the number of cit-
rus, Arabidopsis and rice CCCH genes vary in most sub-
families (Fig. 4). The CCCH-b, c, d and e subfamilies of
citrus contain fewer members (17, 8, 8, 11, respectively)
than the Arabidopsis (20, 8, 11, 13, respectively) and the
rice subfamiles (22, 9, 9, 13, respectively). The CCCH-
c subfamily has a number of members equal to that of
Arabidopsis. However, the citrus CCCH-a subfamily has
the largest number of members among the five subfamilies

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Mol Genet Genomics (2014) 289:855–872 861

Table 2  Statistics on the Group Type Citrus Poplar Arabidopsis Rice Maize

numbers of CCCH motifs for
each CCCH motif class from 1 C–X4–C–X5–C–X3H 0 0 2 0 1
Clementine mandarin (Cle),
2 C–X5–C–X4–C–X3H 8 16 11 9 9
Arabidopsis (At), rice (Os),
Populus (Pt) and maize 3 C–X7–C–X4–C–X3H 7 9 5 8 11
4 C–X7–C–X5–C–X3H 46 77 42 41 45
5 C–X7–C–X6–C–X3H 0 0 1 2 4
6 C–X8–C–X4–C–X3H 1 2 2 1 4
7 C–X8–C–X5–C–X3H 63 96 83 77 98
8 C–X8–C–X6–C–X3H 1 5 1 0 1
9 C–X9–C–X5–C–X3H 3 4 3 6 2
10 C–X10–C–X5–C–X3H 2 0 1 3 0
11 C–X10–C–X6–C–X3H 0 0 0 1 0
12 C–X11–C–X5–C–X3H 1 1 1 1 1
13 C–X11–C–X6–C–X3H 0 1 0 0 0
The CCCH motif types of 14 C–X12–C–X5–C–X3H 0 0 0 0 2
Arabidopsis, rice were adapted
from Wang et al. (2008a). The 15 C–X13–C–X5–C–X3H 0 0 0 0 1
CCCH motif types of Populus 16 C–X15–C–X5–C–X3H 0 0 0 1 0
and maize derived from the 17 C–X17–C–X6–C–X3H 0 0 0 0 1
studies of Chai et al. (2012) and Total C–X4–17–C–X4–6–C–X3H 132 211 152 150 180
Peng et al. (2012), respectively

Fig. 2  Chromosomal locations of citrus CCCH genes. 54 CCCH and CleC3H54) reside on unassembled scaffolds. The length of the
genes are mapped to all 9 chromosomes, while eight CCCH genes chromosome can be estimated using the scale on the left
(CleC3H4, CleC3H26, CleC3H32, CleC3H33, CleC3H34, CleC3H53

(18), which was more than Arabidopsis and rice (15 and CCCH protein sequences (Fig. 3a). The results show that
14, respectively). the number of CCCH motifs in citrus and the spacing amino
To provide further insight into evolutionary relationships, acids between adjacent CCCH motifs is variable, similar to
we constructed five phylogenetic trees using citrus full-length the Arabidopsis and rice CCCH proteins (Wang et al. 2008a).

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862
Fig. 3  Phylogenetic relationships, gene structure, and motif com-
positions of Citrus CCCH genes. a Multiple alignments of 62 full-
length CCCH proteins from citrus were conducted by ClustalX2.1
and the phylogenetic tree was constructed using MEGA4.0 with
1,000 bootstrap replicates. The percentage bootstrap scores higher
than 50 % are indicated on the nodes. b Exon/intron organization of
Functional annotation, gene structure and conserved motif
analysis of citrus CCCH genes
GO annotation of 62 CCCH proteins were performed
using Blast2GO. Based on GO annotation, 61 (CleC3H61
excluded) sequences were assigned GO numbers (Online
13
Mol Genet Genomics (2014) 289:855–872
citrus CCCH genes. Green boxes represent exons and black lines rep-
resents introns. The size of exons and introns can be estimated using
the scale at bottom. c Schematic representation of the conserved
motifs in citrus CCCH proteins. The size of proteins can be estimated
using the scale at the bottom. The name of motifs was listed at the top
the figure (color figure online)
Source 3). For biological functions, metabolic and cellu-
lar processes (24 %) accounted for the largest percentage
among the 61 sequences, followed by single-organism
process and biological regulation (11 %), while the other
7 groups accounted for 30 % out of all the 11 groups. The
binding section of the molecular function category, which
Mol Genet Genomics (2014) 289:855–872 863
includes organic cyclic compound binding, heterocyclic
compound binding, and ion binding, had the largest per-
centage (29 %), followed by small molecular binding
(7 %). With regard to the cellular component, 50 % of the
sequences were assigned to cell part followed by mem-
brane-bounded organelle (35 %). These annotations reveal
that the CCCH genes participate in various biological pro-
cesses and have diverse molecular functions.
To investigate the structural diversity of the citrus CCCH
genes, we compared the exon/intron organization in the
coding sequences of each genes (Fig. 3b). The results are
consistent with the characteristics identified in the above
phylogenetic analyses; most closely related members in the
same subfamilies share similar exon/intron structure, simi-
lar to a previous investigation (Chai et al. 2012). Interest-
ingly, the CCCH genes in subfamily CCCH-d possess no
introns with the exception of CleC3H51, which has two
introns. In contrast, gene structure appears to be more vari-
able among the other four subfamilies. Most subfamilies
have more than two exon/intron structure variants.
Furthermore, we predicted the conserved motifs shared
among related proteins within the family using SMART
online server. We discovered 16 different conserved motifs
(Fig.  3c and Online Source 4), which is greater than the
number found in Arabidopsis (13) and Populus (15) CCCH
proteins (Wang et al. 2008a; Chai et al. 2012). As expected,
most of the closely related members had common motif
compositions within the same family, suggesting functional
similarities. Interestingly, CleC3H13 and CleC3H14, which
may encode nucleocytoplasmic shuttling proteins, contain
two identical C–X8–C–X5–C–X3–H motifs separated by 18
amino acids (Fig. 5a), and therefore were regarded as the
typical tandem zinc finger (TZF) family proteins (Blacks-
hear 2002; Pomeranz et al. 2010b). Previous studies indicate
that TZF proteins can promote target mRNA deadenylation
and degradation via binding to class II ARE elements in the
3′-UTR of target mRNAs (Barreau et al. 2005; Pomeranz
et al. 2010a). Therefore, the two citrus TZF proteins might
have RNA-binding abilities as well. The alignments of 11
TZF proteins from citrus, Arabidopsis, rice and Populus
are shown in Fig. 5a. Phylogenetic analysis revealed that
CleC3H13 and CleC3H14 are the closest homologs to the
Populus counterparts PtC3H17, PtC3H18, and PtC3H20,
suggesting that this type of protein may have a different evo-
lutionary process in woody plants as compared to annual
plants (Fig. 5b). Previous studies indicate that ankyrin repeat
motifs (ANK) may play various roles in diverse molecular
processes, such as transcriptional initiation, ion transpor-
tation and signal transduction (Bork 1993; Sedgwick and
Smerdon 1999; Becerra et al. 2004). Remarkably, five mem-
bers (CleC3H27, 28, 29, 53 and 62) in subfamily CCCH-d
contained two ANK motifs (Figs. 3c, 5c) except two CCCH
motifs (C–X7–C–X5–CX3–H and C–X5–C–X4–C–X3–H).
In addition, RNA recognition motif (RRM) was the sec-
ond largest motif among the 62 CCCH proteins, which are
known to bind single-stranded RNA containing one or more
copies of a putative RNA-binding domain. A total of 14
RRM motifs were identified in 14 CCCH genes, the lengths
of which range from 64 to 97 aa and show little difference
as compared with previous studies (about 90 amino acids)
(Adam et al. 1986; Swanson et al. 1987). Detailed informa-
tion for the 16 motifs is listed in Online Source 4.
To further identify sequence features of the CCCH
motifs, we compared the weblog among these four species,
which performed sequence alignments of 645 CCCH motifs
(132 from citrus, 152 from Arabidopsis, 150 from rice and
211 from Populus) using the Clustal X program. Similar to a
previous study (Wang et al. 2008a), the four (three cysteines
and one histidine) amino acids are highly conserved and
most of them contain glycine and phenylalanine. There is
very little difference amongst the sequence logos derived
from the four plant species (Online Source 5).
Genetic variation of 26 CCCH genes from Poncirus
trifoliata and Fortunella japonica
Based on the whole-genome deep re-sequencing data
(30-fold) of P. trifoliata and F. japonica, a large number
of single nucleotide polymorphisms (SNPs) and inser-
tion/deletions (InDels) were identified in 26 citrus CCCH
genes (including 5′-UTR, 3′-UTR, intron and exon regions)
(Table 3). In P. trifoliata, the five subfamilies (CCCH-a, b,
c, d and e) had 7, 4, 6, 6 and 3 members, respectively, that
contained SNPs and InDels. The number of SNPs ranged
from 14 (CleC3H48) to 130 (CleC3H40) with an average
of 61. The number of SNPs in F. japonica varied from 13
(CleC3H48) to 95 (CleC3H24) with an average of 44. A
total of 1,577 and 1,132 SNPs were obtained from P. trifo-
liata and F. japonica, respectively, as well as 507 common
SNP in the two species. It is noteworthy that the number of
SNPs in P. trifoliata is more than in F. japonica except for
CleC3H03, CleC3H27 and CleC3H52.
The distribution of InDels was also investigated in the
two species. The results show that the number of InDels is
far less than the number of SNPs. In P. trifoliata, a total
of 318 InDels were found, and the number ranges from 4
(CleC3H18 and CleC3H23) to 38 (CleC3H40) with an
average of 12 in the 26 CCCH genes. With regard to the
number of InDels in F. japonica, 220 InDels were obtained
in total, and the number varied from 2 (CleC3H44) to 26
(CleC3H24) with an average of 8. Similarly, most members
contained fewer InDels in F. japonica than in P. trifoliata
with the exception of five members: CleC3H18, 30, 36, 50
and 53. We also noted that the number of shared common
InDels accounts for a smaller proportion than the shared
common SNPs between the two species.
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864 Mol Genet Genomics (2014) 289:855–872

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 Mol Genet Genomics (2014) 289:855–872 865
◂ Fig. 4  Phylogenetic trees of full-length CCCH domain proteins
from Citrus, Arabidopsis, and rice. All CCCH proteins of Citrus (62),
Arabidopsis (68) and rice (67) were divided into five distinct subfam-
ilies (a–e), which were adapted from Chai et al. (2012). The unrooted
tree was constructed based on the full-length protein sequences using
MEGA4.0. The numbers at nodes represent the percentage bootstrap
scores and only bootstrap values higher than 50 % from 1,000 repli-
cates are shown. Citrus CCCH proteins were marked with red dots.
Pentacles behind the CCCH genes represent that these genes were
selected for additional expression pattern investigation. Two pentacles
represent the eight genes that were studied under ABA and drought
stress conditions (color figure online)
Expression patterns of citrus CCCH genes in different
tissues and under various stresses
The expression patterns of 24 CCCH genes were inves-
tigated in various tissues (Figs. 4, 6). Most of the genes
contain CCCH motifs, but some have highly similar gene
structures and conserved motifs or low similarity with
Fig. 5  Alignment analysis of CCCH motif and ANK motif. a Align-
ments of typical TZF genes (including two identical C–X8–C–X5–C–
X3–H motifs separated by 18 amino acids) from Citrus, Arabidopsis,
other un-examined genes, which could serve as reference
for other un-surveyed genes. The majority of CCCH genes
exhibited distinct tissue-specific expression patterns across
all three tissues. Among the 24 CCCH genes, we observed
that 16 genes have the highest transcript accumulation in
young leaves (CleC3H03, 05, 11, 16, 17, 21, 22, 27, 39, 44,
45, 48, 49, 56, 58 and 60) and 7 genes (CleC3H07, 23, 28,
32, 36, 46 and 59) have the highest transcript accumulation
in stems. Only four genes (CleC3H30, 36, 48 and 56) have
relatively high expression in roots. Interestingly, genes
that have the most similar gene structures and conserved
motifs have similar expression patterns in the three tissues,
e.g., genes CleC3H11/CleC3H39, CleC3H16/CleC3H17,
CleC3H21/CleC3H22, but a little difference appeared
between CleC3H45 and CleC3H46. The results indi-
cate that these genes have different expression patterns in
various tissues and those that have similar gene structures
and share conserved motifs may have similar biological
rice, Populus, and human. b Phylogenetic tree of these genes which
contain typical TZF motifs from the five species mentioned above. c
Alignments of ANK from five citrus CCCH genes
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866 Mol Genet Genomics (2014) 289:855–872

Table 3  Statistics on SNPs and Gene name Locus name SNP Indel Subfamily
Indels for 26 CCCH genes of
PT and FJ as compared with PT FJ Common PT FJ Common
Clementine genome using
whole-genome re-sequencing CleC3H03 Ciclev10028282m.g 39 57 19 8 7 0 a
data CleC3H11 Ciclev10015789m.g 66 39 16 15 6 1 c
CleC3H16 Ciclev10004417m.g 41 34 11 14 9 1 e
CleC3H17 Ciclev10007946m.g 66 40 17 6 5 1 e
CleC3H18 Ciclev10014902m.g 55 41 22 4 5 2 a
CleC3H19 Ciclev10007876m.g 62 35 13 15 8 0 a
CleC3H23 Ciclev10008089m.g 69 31 11 4 3 0 a
CleC3H24 Ciclev10027798m.g 126 95 52 28 26 3 b
CleC3H27 Ciclev10030920m.g 40 43 21 5 5 3 d
CleC3H28 Ciclev10004423m.g 65 31 16 13 8 1 d
CleC3H29 Ciclev10030816m.g 58 41 21 23 13 6 d
CleC3H30 Ciclev10018660m.g 99 89 40 20 21 7 c
CleC3H34 Ciclev10031666m.g 78 59 29 25 10 3 e
CleC3H35 Ciclev10012360m.g 33 21 10 13 6 0 c
CleC3H36 Ciclev10016092m.g 102 75 35 7 9 1 c
CleC3H39 Ciclev10008851m.g 51 35 11 7 5 1 c
CleC3H40 Ciclev10014173m.g 130 88 39 38 20 9 b
CleC3H43 Ciclev10030728m.g 64 39 17 20 16 0 a
CleC3H44 Ciclev10018237m.g 43 20 9 5 2 0 a
CleC3H48 Ciclev10008633m.g 14 13 6 9 5 1 d
CleC3H50 Ciclev10021165m.g 52 39 18 10 11 2 b
Common: Identical SNP CleC3H52 Ciclev10014515m.g 53 65 26 13 11 4 a
and Indel from CCCH genes
between P. trifoliata and CleC3H53 Ciclev10014437m.g 55 45 27 8 10 2 d
F. japonica. Subfamily: CleC3H59 Ciclev10010506m.g 95 55 28 9 9 1 b
These genes belong to CleC3H60 Ciclev10007413m.g 96 80 32 10 5 3 c
which subfamily divided by CleC3H62 Ciclev10027883m.g 33 32 14 10 6 0 d
phylogenetic relationship
Total 1,577 1,132 507 318 220 46
PT, P. trifoliata; FJ, F. japonica

functions. Detailed information for the 24 pairs of gene-
specific primers is listed Online Source 6.
Subsequently, the expression profiles of eight CCCH
genes were further analyzed under two stress conditions,
mannitol and ABA (Fig. 7a). Genes CleC3H05, 44, 56 con-
tain both CCCH and RRM motifs while genes CleC3H11,
16, 21, 48 and 60 contain only CCCH motifs (1–3). These
CCCH genes showed high expression in leaves. All the
selected genes reflected different relative expressions at dif-
ferent time points against different stress treatments. For
detailed information on expression level differences, refer
to Online Source 7.
Under drought stress, the results showed that the
CleC3H05 gene was up-regulated significantly at all time
points after treatment (Fig. 7a). By contrast, the rela-
tive expression of four genes (CleC3H16, 48, 56, and 60)
increased obviously at most time points, while CleC3H44
was up-regulated significantly only at 1 h after treatment
and CleC3H21 was up-regulated at 24 and 48 h after
treatment. In addition, we also examined the expression
patterns of eight genes under ABA stress conditions. As
shown in Fig. 7b, the expression of CleC3H05 was greatly
up-regulated during the whole process, while CleC3H11
and CleC3H56 displayed significantly higher expression at
most time points after treatment. In comparison, CleC3H44
and CleC3H48 were obviously down-regulated at most
time points, while CleC3H16, CleC3H21 and CleC3H60
did not show any obvious variation.
Ultimately, among these selected genes, CleC3H05
demonstrated a significantly positive response to both
drought and ABA stresses at all time points after treat-
ment. In contrast, CleC3H16, CleC3H21, CleC3H48 and
CleC3H56 show positive responses to drought stress at
some time points, while only CleC3H11 and CleC3H56
show obvious positive responses to ABA stress. The anal-
ysis of expression patterns of these CCCH genes may pro-
vide the basis for further study of the functions of these
proteins in citrus.

13
Mol Genet Genomics (2014) 289:855–872 867

Fig. 6  The expression patterns of 24 selected genes in various citrus expression level. R, S, and L represent root, stem and leaf, respec-
tissues. Quantitative real-time PCR was used to analyze the 24 spe- tively. Bars are standard deviations from at least three technical
cific gene expression patterns. Y-axis represents the mRNA relative repeats

13

868
Fig. 7  The expression profiles of eight selected genes in citrus after
treatments. a The eight specific gene expression patterns at six time
points after drought treatment. b The eight selected gene expression
patterns at six time points after ABA treatment. The y-axis represents
Discussion
The CCCH-type zinc finger proteins are specific and dis-
tinct from other zinc finger transcription factors because
they regulate gene expression by binding to mRNA. CCCH
domain-containing proteins have been implicated in multi-
ple biological processes such as plant growth, development,
and environmental responses. Previous studies have inves-
tigated and analyzed the features and functions of CCCH
genes in plants such as Arabidopsis, rice, maize, Populus,
and Medicago truncatula (Wang et al. 2008a; Chai et al.
2012; Peng et al. 2012; Zhang et al. 2013). In this study,
we identified 62 non-redundant citrus CCCH genes by
genome-wide analysis. This is the first comprehensive anal-
ysis of CCCH genes in citrus, a species which has not expe-
rienced recent whole-genome duplication (WGDs) events.
We utilized phylogenetic analyses to better understand the
potential biological functions of the citrus CCCH genes
during evolutionary processes. A bioinformatics analysis
suggests that the CCCH gene family contains diverse gene
structures and conserved motifs. Large numbers of variants
13
Mol Genet Genomics (2014) 289:855–872
the mRNA relative expression level; number on the x-axis represents
different time points after treatment. Bars are standard deviations
from at least three technical repeats
identified in two other important citrus genera offer valu-
able resources for further genomics and genetics research.
The expression profiles of many CCCH genes in different
tissues and under various stress conditions show that they
play important roles in citrus development and environ-
mental responses.
As shown in Table 2, two conventional CCCH motifs
of C–X7–8–C–X5–C–X3–H accounted for the major-
ity (82.4 %) of all citrus CCCH motifs, similar to those
found in Arabidopsis (82.2 %), rice (78.8 %), and Popu-
lus (82.0 %). This suggests that the CCCH motifs in citrus
are also highly conserved (Chai et al. 2012). It is notewor-
thy that the C–X7–C–X6–C–X3H motif has been identified
in Arabidopsis, rice, and maize but is absent in citrus and
Populus, which suggests that this motif did not play any
functional role in woody plants and therefore has been lost
during the evolutionary processes. Moreover, the num-
ber of CCCH genes identified in citrus (62) is less than in
Arabidopsis (68), which is inconsistent with the threefold
larger genome size of the Clementine mandarin (367 Mb)
(Hamon et al. 2003) versus that of Arabidopsis (125 Mb).
Mol Genet Genomics (2014) 289:855–872 869
This finding is similar to another analyzed MADS-Box
gene family (Hou et al. 2014). Gene duplication events,
including segmental and tandem duplications, are a pri-
mary driving force throughout the expansion and evolu-
tion of gene families (Moore and Purugganan 2003). The
model plants Arabidopsis, rice, and Populus have under-
gone recent duplication events, which lead to the large-
scale expansion of gene families in their genomes (Tay-
lor and Raes 2004; Tuskan et al. 2006). Previous studies
have shown that there were no WGDs in citrus except an
ancient triplication, called the γ event, which was shared
by all core eudicots (Jiao et al. 2011; Xu et al. 2013). In
addition, there is only one example of tandemly arrayed
CCCH genes in the same chromosomal location, which is
far less than those found in the plants that have undergone
recent duplication events (Fig. 2). Xu et al. (2013) previ-
ously showed that citrus is rather ancient and evolutionarily
close to cacao. An infrequent reproductive cycle in some
taxa because of apomixis, male or female sterility, long
juvenility, and vegetative propagation may contribute to the
restriction of genome expansion and evolution. Therefore,
recent WGDs lead to a greater number of CCCH genes in
Arabidopsis and rice than in citrus. The greater number of
CCCH genes and motifs found in Populus as compared to
citrus may not be due only to the larger Populus genome
size but also to two rounds of WGDs and one tandem
duplication event in Populus (Tuskan et al. 2006; Yang
et al. 2008; Chai et al. 2012).
The recent segmental duplication occurs most frequently
in plants because most plants are diploidized polyploids
and retain numerous duplicated chromosomal blocks in
their genomes (Blanc and Wolfe 2004). The long-term
evolutionary fate of duplicated genes is determined by
the functions of the duplicated genes. There are four main
types of functional differentiation by gene duplication:
pseudogenization, conservation of gene function, neo-
functionalization, and subfunctionalization (Zhang 2003).
Many duplicated genes may be lost after the duplication
events, but subfunctionalization and neofunctionalization
are the primary factors for the retention of new genes. With
regard to neofunctionalization of duplicated genes, posi-
tive selection contributes to advantageous mutations that
strengthen the ability of organisms to respond to various
environmental conditions. In the case of subfunctionaliza-
tion of duplicated genes, each daughter gene will inherit at
least one function of the ancestral gene and can refine its
functions through further substitutions under positive selec-
tion (Taylor and Raes 2004). Therefore, duplicated genes
play important roles in plants that experience WGD events.
However, due to the absence of gene duplication events in
the CleC3Hs family, the functions of CleC3Hs genes are
probably more conservative than AtC3Hs, OsC3Hs and
PtC3Hs. In addition, the CleC3Hs genes likely experienced
more advantageous mutations and developed new functions
during evolutionary processes.
A integrative phylogenetic tree using the three repre-
sentative species (citrus, Arabidopsis and rice) could not be
constructed due to some genes have low bootstrap values.
Therefore, we divided the genes into five subfamilies based
on a previous study (Chai et al. 2012). The citrus CCCH-
a subfamily contains the largest number of genes among
the five subfamilies (18), which is more than Arabidopsis
and rice (15 and 14, respectively). The other four subfami-
lies have fewer genes (Fig. 4). These results may suggest
that the CCCH gene duplication event mainly happened in
CCCH-b, c, d and e subfamiles after the ancient triplica-
tion shared by all core eudicots (Jiao et al. 2011). A pre-
vious study demonstrated that citrus is phylogenetically
closer to Arabidopsis than to rice (Xu et al. 2013). It was
estimated that citrus and Arabidopsis diverged 85 million
years ago (MYA), hereafter, an additional two rounds of
recent WGD occurred in Arabidopsis, called the α and β
events. However, the diverged time between citrus and rice
was estimated to be 115 MYA before the γ event (Jiao et al.
2011; Xu et al. 2013). The results based on Fig. 4 dem-
onstrate similar evidence, such as the finding that almost
all CleC3Hs show closer relationships than OsC3Hs with
AtC3Hs, which suggests that orthologous genes between
dicots have a closer relationship than orthologous genes
between monocots and dicots. In addition, further align-
ments of amino acid sequences based on stringent criteria
revealed that there are far fewer paralogous genes in the
CCCH gene family in citrus than in Arabidopsis and rice.
This suggests that CCCH gene duplication in Arabidopsis
and rice was more prevalent than in citrus. Furthermore,
we constructed five subfamily phylogenetic trees using
62 CCCH proteins and analyzed the gene structures and
conserved motifs (Fig. 3). Remarkably, the CCCH genes
belonging to the same subfamily always displayed simi-
lar gene structure and domain architecture, suggesting
that these genes may have similar functions. However, we
found that many genes have different structures and con-
served motifs, suggesting that the 62 CCCH genes have
diverse biological functions and molecular functions. A
Blast2GO annotation supports this hypothesis.
SNPs are the most abundant genomic DNA variations
and are ubiquitous in both functional genes and non-coding
regions (Brookes 1999). They are conserved during evo-
lution, associated with genetic traits, and suited for high
throughput genotyping. SNPs have increasingly become a
popular and powerful tool for various genetics and genom-
ics studies such as the mapping of whole genomes, tag-
ging of important traits, comparison of genome evolution,
and classification of diverse clades (Rickert et al. 2003;
Han et al. 2009). Poncirus trifoliata and F. japonica are
two important species that belong to the Poncirus and
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870
Fortunella genera, respectively. In this study, we obtained a
large number of SNPs and InDels in 26 citrus CCCH genes
based on re-sequencing data from the two species. The
results indicate that P. trifoliata has a more distant genetic
relationship with Clementine mandarin than F. japonica,
which is consistent with previous studies (Nicolosi et al.
2000; Liu et al. 2013). These variants could greatly contrib-
ute to the functional diversity of CCCH genes, which may
be involved in the acclimation of plants to various envi-
ronments during evolutionary processes. In addition, the
CCCH genes that contain large numbers of variants may be
highly conserved and retained after they experienced posi-
tive selection.
Evidence is accumulating that the CCCH genes are
involved in plant development and stress responses. Inter-
estingly, almost all of these functionally characterized
CCCH genes belong to either the CCCH-b (1–6 C–X8–C–
X5–C–X3–H) or the CCCH-d (1 C–X7,8,10–C–X5–C–X3–H
and 1 C–X5–C–X4–C–X3–H) subfamilies. For example,
AtC3H14 and AtC3H15 (secondary cell wall biosynthesis)
and AtC3H37 (floral morphogenesis) belong to the CCCH-b
subfamily, while AtC3H2 (seed germination) and AtC3H54
(embryo development) belong to the CCCH-d subfamily in
Arabidopsis (Li and Thomas 1998; Li et al. 2001; Kim et al.
2008; Ko et al. 2009). In addition, OsC3H12 (disease resist-
ance) in rice and CsEF1 (embryo development) in cucumber,
and OsC3H2 (salt response) and GHZFP1 (biotic and abi-
otic stresses) in cotton, belong to the CCCH-b and CCCH-d
subfamilies, respectively (Grabowska et al. 2009; Guo et al.
2009; Deng et al. 2012; Jan et al. 2013). However, there are
very few reports regarding CCCH genes in citrus. Therefore,
we investigated the temporal and spatial expression patterns
of many CCCH genes from the five subfamilies. Firstly, we
selected 24 CCCH genes for expression pattern elucidation in
different tissues (Fig. 6). These genes have different expres-
sion patterns; most of them show high levels of expression in
leaves, but a small portion has the highest relative expression
in stem tissue. Only a few genes have higher expression lev-
els in root than in the leaves or shoot. In addition, to obtain
insight into the stress responses of some CCCH genes, the
expression profiles of eight CCCH genes were investigated
under two stress conditions: drought and ABA (Fig. 7). The
results indicate that the expression of CleC3H11, 16, 21, 48
(contain only CCCH motif), and CleC3H56 (contain both
RRM and CCCH motifs) is up-regulated significantly at
most time points under drought or ABA stress conditions.
Most importantly, CleC3H05 (2 CCCH and 1 RRM, CCCH-
e) has a dramatically positive response to both drought and
ABA stress conditions. This may suggests that genes con-
taining both RRM and CCCH motifs can play roles better.
Besides, the un-surveyed gene CleC3H04 shares similar
gene structure and conserved motifs with CleC3H05, sug-
gesting that they may have similar functions. These surveyed
13
Mol Genet Genomics (2014) 289:855–872
genes belong to different subfamilies, which suggests that
many genes also play crucial roles in response to stress
conditions except genes in the CCCH-b and CCCH-d sub-
families. The different expression patterns of the 24 CCCH
genes in different tissues and 8 CCCH genes under the two
stress conditions may provide insight to further investigate
the roles of these candidate genes in citrus. Moreover, other
CCCH genes may also play important roles under various
stress conditions and need to be further analyzed.
In conclusion, we have completed a genome-wide iden-
tification and comprehensive analysis of the CCCH zinc
finger genes in citrus. The results demonstrate the impor-
tance of CCCH zinc finger genes in the regulation of plant
development and in responses to various biotic and abiotic
stresses. This information provides valuable resources for
further evolutionary, genetic, and functional studies of the
CCCH gene family in both citrus and in other plant species.
Acknowledgments  We appreciate the editor and the reviewers
for their constructive suggestions and comments. This research was
supported financially by the National Natural Science Foundation of
China (Grant Nos. 31130046, 31372046 and 31071777). We acknowl-
edge the International Citrus Genome Consortium for using the Clem-
entine genome sequences, and other institutions and organizations
for providing the public release of genome sequences used in our
investigation. We also appreciate Zhongchi Liu and Rachel Maczis
Shahan for their support and comments during the preparation of this
manuscript.
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