Published March 11, 2016
o r i g i n a l r es e a r c h
Genome-Wide Identification of the AP2/ERF
Gene Family Involved in Active Constituent
Biosynthesis in Salvia miltiorrhiza
A. J. Ji, H. M. Luo, Z. C. Xu, X. Zhang, Y. J. Zhu, B. S. Liao, H. Yao,
J. Y. Song,* and S. L. Chen*
Abstract
Tanshinones and phenolic acids are the major bioactive constituents in the traditional medicinal crop Salvia miltiorrhiza; however,
transcription factors (TFs) are seldom investigated with regard to
their regulation of the biosynthesis of these compounds. Here a
complete overview of the APETALA2/ethylene-responsive factor
(AP2/ERF) transcription factor family in S. miltiorrhiza is provided,
including phylogeny, gene structure, conserved motifs, and gene
expression profiles of different organs (root, stem, leaf, flower)
and root tissues (periderm, phloem, xylem). In total, 170 AP2/ERF
genes were identified and divided into five relatively conserved
subfamilies, including AP2 (25 genes), DREB (61 genes), ethylene
responsive factor (ERF; 79 genes), RAV (4 genes), and Soloist (1
gene). According to the distribution of bioactive constituents and
the expression patterns of AP2/ERF genes in different organs and
root tissues, the genes related to the biosynthesis of bioactive
constituents were selected. On the basis of quantitative real-time
polymerase chain reaction (qRT-PCR) analysis, coexpression
analysis, and the prediction of cis-regulatory elements in the promoters, we propose that two genes (Sm128 and Sm152) regulate
tanshinone biosynthesis and two genes (Sm008 and Sm166)
participate in controlling phenolic acid biosynthesis. The genes
related to tanshinone biosynthesis belong to the ERF-B3 subgroup. In contrast, the genes predicted to regulate phenolic acid
biosynthesis belong to the ERF-B1 and ERF-B4 subgroups. These
results provide a foundation for future functional characterization
of AP2/ERF genes to enhance the biosynthesis of the bioactive
compounds of S. miltiorrhiza.
Published in Plant Genome
Volume 9. doi: 10.3835/plantgenome2015.08.0077
© Crop Science Society of America
5585 Guilford Rd., Madison, WI 53711 USA
This is an open access article distributed under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
the pl ant genome
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alvia miltiorrhiza Bunge, a well-known traditional
Chinese medicine, is widely used to treat cardiovascular diseases in Asia, the United States, and some
European countries; the medicine exhibits strong antidementia, neuroprotective, antioxidant, and anti-inflammatory activities (Zhou, Zuo, et al., 2005; Hügel and
Jackson, 2014). According to their chemical properties,
the medicinal active constituents of S. miltiorrhiza are
divided into two groups: hydrophilic compounds (e.g.,
salvianolic acid A, salvianolic acid B, Danshensu) and
lipophilic compounds (e.g., tanshinone I, IIA, and IIB;
cryptotanshinone) (Wang, Morris-Natschke et al., 2007).
In the past 20 yr, many genes that encode key enzymes
in the biosynthesis pathways of these active compounds
have been cloned and functionally analyzed (Huang et
al., 2008a; Huang et al., 2008b; Liao et al., 2009; Xiao
et al., 2009; Yan et al., 2009; Kai et al., 2010; Kai et al.,
2011; Song and Wang, 2011; Cui et al., 2015). Genomic
and transcriptomic studies of S. miltiorrhiza have also
A.J. Ji, H.M. Luo, Z.C. Xu, X. Zhang, B.S. Liao, H. Yao, and J.Y.
Song, Inst. of Medicinal Plant Development, Chinese Academy
of Medical Sciences and Peking Union Medical College, Beijing
100193, China; J. Y. Song, Chongqing Institute of Medicinal Plant
Cultivation, Chongqing 408435, China; Y. J. Zhu and S.L. Chen,
Inst. of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China. Received 27 Apr. 2015.
Accepted 18 Oct. 2015. *Corresponding authors (jysong@implad.
ac.cn; slchen@icmm.ac.cn).
Abbreviations: ADS, amorpha-4, 11-diene synthase; ALDH1, aldehyde dehydrogenase 1; AP2/ERF, APETALA2/ethylene-responsive
factor; cDNA, complementary DNA; DREB, dehydration-responsive
element binding proteins; ERF, ethylene responsive factor; GT, glandular trichome; NJ, neighbor-joining; qRT-PCR, quantitative real-time
polymerase chain reaction; RAV, related to ABI3/VP1; SRA, Sequence Read Archive; TF, transcription factor.
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progressed rapidly (Ma et al., 2012; Luo et al., 2014;
Wang et al., 2014; Zhang et al., 2015). In particular, the
genome of S. miltiorrhiza has been completely assembled
(SRA [Sequence Read Archive] accession: SRP051524).
Increased knowledge regarding the functional genomics
and physiological properties of S. miltiorrhiza has made
it an ideal model medicinal plant (Shao and Lu, 2013;
Li and Lu, 2014). Compared with the great progress in
cloning the enzymes of the synthetic pathway of active
compounds in S. miltiorrhiza, little is known about the
mechanisms by which TFs regulate tanshinone and phenolic acid biosynthesis (Zhang et al., 2013).
The AP2/ERF TF family, one of the largest families
of TFs in the plant kingdom, covers five subfamilies: AP2
(APETALA2), ERF (ethylene-responsive factor), DREB
(dehydration-responsive element binding proteins),
RAV (related to ABI3/VP1), and Soloist ( Sakuma et al.,
2002; Yamasaki et al., 2013; Licausi et al., 2013). This TF
family has been reported to be involved in secondary
metabolism, and the members all belong to the ERF-B3
subgroup. Moreover, several AP2/ERF TFs function as
core regulators that positively regulate the biosynthesis
of artemisinin (Yu et al., 2012; Lu et al., 2013), which
is a terpenoid. ERF-B3 proteins interact with the GCC
box element of target genes and regulate their expression. This regulation affects the biosynthesis and accumulation of secondary metabolites (Ohme-Takagi and
Shinshi, 1995; Sears et al., 2014). Identification of GCC
box elements in promoters of pathway genes may help
to confirm the interaction between AP2/ERF genes and
the pathway genes. AP2/ERF TFs also regulate alkaloids
in Catharanthus roseus (Menke et al., 1999; van der Fits
and Memelink, 2001) and Nicotiana tabacum (Shoji et al.,
2010). Therefore, we proposed that AP2/ERF TFs may be
involved in tanshinone and phenolic acid biosynthesis.
The genome-wide identification of AP2/ERF TFs has
been documented in several plants such as Arabidopsis thaliana (Sakuma et al., 2002; Nakano et al., 2006),
Oryza sativa (Nakano et al., 2006), maize (Zhuang et al.,
2010), Glycine max (Zhang et al., 2008), Prunus mume
(Du et al., 2013), Brassica rapa ssp. pekinensis and Vitis
vinifera ( Zhuang et al., 2009; Licausi et al., 2010; Song
et al., 2013). In this study, we performed a genome-wide
survey and a systematic characterization of the AP2/ERF
family. In addition, the expression patterns of AP2/ERF
genes in different organs (root, stem, leaf, flower) and
root tissues (periderm, phloem, xylem) were revealed by
means of RNA-seq data and qRT-PCR detection. Furthermore, using the phylogenetic tree and coexpression
analyses, we proposed four candidate AP2/ERF genes
that may be involved in regulating tanshinone and phenolic acid biosynthesis. Overall, systematic analysis of
the AP2/ERF family will provide a new gene resource for
the metabolic engineering of the secondary metabolites
of S. miltiorrhiza.
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MATERIALS AND METHODS
Plant Materials and Treatment
In this study, S. miltiorrhiza Bunge (line 99-3), which
was cultivated in the experimental field at the Institute of
Medicinal Plant Development (China), was used. Roots,
stems, leaves, flowers, periderm, phloem, and xylem were
collected to analyze the expression patterns of AP2/ERF
genes. All of the sample materials were immediately frozen in liquid nitrogen and stored at -80°C.
Database Search and Gene Identification
The AP2/ERF sequences of A. thaliana were downloaded
from the Arabidopsis Information Resource (http://Arabidopsis.org/). A search of the S. miltiorrhiza genome
database (SRA accession: SRP051524) was conducted to
obtain all AP2/ERF family members. The AP2-domain
hidden Markov model (HMM: PF00847) was used to
screen the S. miltiorrhiza genome database for all of the
gene sequences that are homologous to AP2/ERF. The
predicted genes were corrected manually by comparison
with other plant AP2/ERFs by means of the BLASTx
algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). All
AP2/ERF protein sequences were then confirmed with
PROSITE (http://prosite.expasy.org/), and the conserved
AP2 domains of each family member were obtained. The
sequence features of the conserved AP2 domains were
analyzed by alignment by means of DNAMAN software.
Phylogenetic, Gene Structure, and Conserved
Motif Analyses
The AP2-domain amino acid sequences of S. miltiorrhiza
and A. thaliana were aligned with MEGA 5.0 software.
Neighbor-joining (NJ) tree construction, gene structures
analysis, and conserved motif detection were performed
as described in a previous study (Xu et al., 2013). The
orthologous proteins were obtained by BLAST analysis
of the candidate AP2/ERF TFs against the A. thaliana
genome. Synteny analysis was performed by comparing
the homology and order of the genes between S. miltiorrhiza scaffolds and A. thaliana chromosomes. The
BLASTp cutoff was set as E-value < 1× 10−10.
Gene Expression Analysis
and Cis-element Prediction
The RNA-seq reads from two replicates of four organs
(root, stem, leaf, flower) and three replicates of three
tissues (periderm, phloem, xylem) were generated by
the Illumina HiSeq 2000 and 2500 platforms, respectively (SRA accessions: SRR1640458, SRP051564, and
SRP028388). The expression patterns of the AP2/ERF
genes in the different organs (root, stem, leaf, flower) and
root tissues (periderm, phloem, xylem) were analyzed
with TopHat and Cufflinks (Trapnell et al., 2012).
The 1500-bp promoter sequences upstream of the
transcription start site (ATG) of 72 and 29 genes encoding
key enzymes involved in the biosynthesis of tanshinones
and phenolic acids, respectively, were obtained from
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whole-genome sequences. The cis-elements targeted by
AP2/ERF TFs were investigated with the PLACE database
(http://www.dna.affrc.go.jp/PLACE/signalscan.html).
RNA Isolation and qRT-PCR
Total RNA was isolated from each sample with an RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China)
according to the manufacturer’s instructions. The quality
and integrity of the RNA were analyzed by electrophoresis and a NanoDrop 2000C spectrophotometer. The
first cDNA strand was synthesized using a FastQuant RT
Kit (Tiangen Biotech). The specific primers for qRT-PCR
were designed with Primer Premier 6.0, and the amplicon
length was set between 130 and 180 bp (Table S1). The
qRT-PCR reagents, instrument, and method were identical
to those in our previous study (Luo et al., 2014). SmActin
was used as the reference gene (Yang et al., 2010). Three
key genes for enzymes—SmDXS2 (1-deoxy-D-xylulose
5-phosphate synthase 2), SmCPS1 (copalyl diphosphate
synthase 1), and SmRAS1 (rosmarinic acid synthase 1)—
were tested as positive controls. Each sample was tested in
triplicate. To detect expression differences of the candidate
genes among various tissues, one-way ANOVA was performed using IBM SPSS 20 software. P < 0.01 was considered highly significant. Gene coexpression analysis of the
candidate genes was performed by means of Pearson’s correlation test, and candidate genes with the Pearson coefficients greater than 0.8 were considered to be coexpressed.
RESULTS
Identification and Phylogenetic Analysis
of the AP2/ERF Family in S. miltiorrhiza
In total, 170 AP2/ERF genes in S. miltiorrhiza that encode
at least one AP2 domain were identified. The lengths of
the genomic DNA, coding sequence, and protein; the AP2
domain sequence; and the number of introns are summarized in Table S2. According to the number and sequence
features of the AP2 domains, the AP2/ERF family of
S. miltiorrhiza was divided into five subfamilies—AP2,
DREB, ERF, RAV, and Soloist—and these subfamilies had
25, 61, 79, 4, and, 1 members, respectively (Fig. S1). The
DREB and ERF subfamilies were the most dominant in S.
miltiorrhiza. Based on the similarities of the AP2-domain
amino acid sequences, the 140 members of the largest two
subfamilies were further assigned to 12 subgroups: DREB
(A1-A6) and ERF (B1-B6). A multiple sequence alignment
between S. miltiorrhiza and A. thaliana was performed
using the AP2 domain sequences from each subfamily, and it indicated that 31, 34, 70, 86, and 59 conserved
amino acid residues were found in the DREB, ERF, AP2,
RAV, and Soloist subfamilies, respectively (Fig. S2).
An unrooted phylogenetic tree was constructed to
confirm these classifications and to analyze their phylogenetic relationships based on the alignments of the AP2
domain sequences of S. miltiorrhiza and A. thaliana (Fig.
1). The analysis showed that the tree was separated into 10
clades, including the AP2, DREB, ERF, RAV, and Soloist
subfamilies. Clade 1 and clade 2 represented the AP2 and
Soloist subfamilies, respectively. Clades 3 and 4 represented
the DREB subfamily, and clade 6 represented the RAV subfamily. Clades 5 and 7–10 indicated the ERF subfamily.
To confirm the subgroups of the DREB/ERF subfamilies, we constructed a deep phylogenetic tree using
the AP2 domain sequences of the DREB/ERF subfamilies
in S. miltiorrhiza and V. vinifera (Fig. S3) because the
AP2/ERF members have been well classified in V. vinifera
(Zhuang et al., 2009; Licausi, et al., 2010). The classification of the DREB/ERF subgroups of S. miltiorrhiza was
consistent with that of V. vinifera.
Gene Structure and Conserved Motifs of SmAP2s
Gene structure was analyzed using the online Gene
Structure Display Server, and the exon-intron structures
are shown in Figures 2 and S4. The number of introns in
the AP2/ERF genes varied among the subfamilies. The
AP2 and Soloist subfamilies contained the most introns
(5 to 10), whereas most DREB/ERF and RAV subfamily members possessed no introns. The positions of
introns also differed among the subfamilies. Fewer than
three introns were found within the AP2-R1 or AP2-R2
domain of the AP2 subfamily. The two DREB members
both had one intron before the conserved AP2 domain.
Most intron positions in the ERF subfamily were identical to those in the DREB subfamily.
Some conserved motifs outside the AP2 domain may
be involved in nuclear localization, transcription activity,
and protein-protein interactions (Ikeda and Ohme-Takagi,
2009; Causier et al., 2012; Tiwari et al., 2012). We therefore
investigated the conserved motifs of all AP2/ERF proteins
in S. miltiorrhiza, and the results are shown in Figures
2 and S4. Twenty-six conserved motifs were identified,
and most members in the same subgroup possessed one
or more motifs together outside the AP2 domain (Table
S3). Motifs 1–8, 10, and 20 corresponded to the AP2
domain region, and motifs 21 and 23 corresponded to the
B3 domain region in the RAV subfamily. The remaining
14 motifs were selectively distributed among particular
subfamilies and subgroups, illustrating the sequence similarity among proteins in the same group. For example,
the AP2 subfamily contained three motifs (9, 16, 26), and
these motifs were not observed in the other subfamilies. Motifs 14, 15, 17 18, 24, and 25 were specific to the
ERF subfamily. Among these motifs, motif 17 was only
observed in the B3 subgroup, and motif 15 was shared by
both B5 and B6 members. The proteins within a subgroup
that shared the same motifs may have similar functions.
Expression Pattern of SmAP2s in S. miltiorrhiza
Tanshinones and phenolic acids, the major bioactive
compounds of S. miltiorrhiza, are distributed primarily
in the roots (Ma et al., 2012; Di et al., 2013). Our previous
study showed that the highest tanshinone content is in
the periderm followed by the phloem and that the lowest
content is in the xylem (Xu et al., 2015). In contrast, the
highest phenolic acid content is in the phloem and xylem,
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Fig. 1. An unrooted phylogenetic tree was constructed with the AP2/ERF (APETALA2/ethylene-responsive factor) domain amino acid
sequences of S. miltiorrhiza and A. thaliana. A neighbor-joining tree was constructed using MEGA5 with 1000 bootstrap replicates.
The tree was divided into 10 groups that contained the AP2, DREB (dehydration-responsive element binding proteins), ERF, RAV (related
to ABI3/VP1), and Soloist subfamilies. The photo in the center of phylogenetic tree presents the plant Salvia miltiorrhiza.
and the lowest content is in the periderm. Moreover, previous studies have also revealed that the expression patterns
of the TFs that regulate secondary metabolism pathways
are similar to the expression patterns of key enzymes and
the distribution of secondary metabolites in A. annua (Yu
et al., 2012; Lu et al., 2013). We therefore investigated the
expression profiles of the AP2/ERF family obtained from
seven sources of transcriptome data, including libraries
from different organs (root, stem, leaf, flower) and root
tissues (periderm, phloem, xylem) of S. miltiorrhiza (Table
S4). Heat maps were also constructed (Fig. 3 and Fig. S5),
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and they showed the different expression patterns of all
AP2/ERF genes across the various organs and tissues.
Excluding the genes that could not be detected in
the transcriptome analyses, the expression of 120 AP2/
ERF genes in at least one of four organs and three tissues
was detected, including 14 genes in the AP2 subfamily,
47 genes in the DREB subfamily, 55 genes in the ERF
subfamily, 3 genes in RAV subfamily, and the 1 Soloist
gene (Table S4). Among these genes, 38, 8, 1, 12, 12, 21,
and 18 AP2/ERF genes were expressed relatively highly in
the root, stem, leaf, flower, periderm, phloem, and xylem,
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Fig. 2. The features of the gene structures and the distribution of conserved motifs of the ethylene responsive factor (ERF) subfamily of S.
miltiorrhiza. (A) Phylogenetic relationships among ERF subgroups. (B) The distribution of the conserved motifs of each ERF protein. (C)
Exon/intron structures of the ERF genes of S. miltiorrhiza.
respectively. Thirty-four of the 38 genes in the root belong
to the DREB and ERF subfamilies, while 11 of the 12 genes
in periderm are ERF members. Interestingly, 26 genes
that were markedly expressed in phloem and xylem are
also members of the DREB and ERF subfamilies. In total,
expression profiling provided a valuable resource for the
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Fig. 3. The expression profiles of S. miltiorrhiza AP2/ERF (APETALA2/ethylene-responsive factor) genes in the root, stem, leaf, and
flower. (A), (B), (C) and (D) indicate the expression patterns of the AP2, ERF, DREB (dehydration-responsive element binding proteins),
and RAV (related to ABI3/VP1)-Soloist subfamilies, respectively. The color bar at the top represents the expression values. The genes in
white were not expressed in these four organs, and the values for exon model per million mapped reads were zero.
identification of candidate genes involved in the regulation
of bioactive compound biosynthesis in S. miltiorrhiza.
Identification of Candidate SmAP2s Genes
Related to Tanshinone Biosynthesis
On the basis of the RNA-seq data of the different organs
and tissues, 11 SmAP2s genes (Sm021, Sm026, Sm032,
Sm082, Sm086, Sm108, Sm109, Sm128, Sm139, Sm152,
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and Sm168) were highly expressed in root and periderm
and were identified as potential regulatory factors related
to tanshinone biosynthesis. Using further analysis with
qRT-PCR (Fig. S6), we concluded that Sm026, Sm082,
Sm086, Sm108, Sm128, and Sm139 showed significant
expression in the root. Sm026, Sm108, Sm128, Sm139,
and Sm152 showed the highest expression in the periderm tissue. The expression of these five genes showed a
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Fig. 4. Expression patterns of selected AP2/ERF (APETALA2/ethylene-responsive factor) genes related to tanshinone (A) and phenolic
acid (B) biosynthesis. Relative expression of the selected AP2/ERF genes in R (root), S (stem), L (leaf), F (flower), R1 (periderm), R2
(phloem), and R3 (xylem) was examined by quantitative real-time polymerase chain reaction. The characters on the x axis indicate different organs and tissues. The y axis shows the relative level of gene expression. The S. miltiorrhiza actin gene was used as an internal
reference. SmDXS2, SmCPS1, and SmRAS1 were tested as positive controls. We performed a one-way ANOVA with IBM SPSS 20 software. Asterisks represent significant differences. P < 0.01 was considered highly significant.
similar pattern with the control terpenoid synthase genes
(SmCPS1 and SmDXS2) (Fig. S6).
Moreover, phylogenetic tree and gene coexpression analyses were performed to narrow the candidate
genes, which were most likely to be related to secondary
metabolism. We observed the phylogenetic relationships
using the AP2 domain sequences of the candidate TFs
and the functional TFs associated with secondary metabolism—ORCA2, ORCA3, AaERF1, AaERF2, NtERF32,
NtERF189, NtORC1, and NtJAP1 (Fig. S7) (De Sutter,
Vanderhaeghen, et al., 2005, Lu, Zhang, et al., 2013,
Menke, Champion, et al., 1999, Sears, Zhang, et al., 2014,
Shoji, Kajikawa, et al., 2010, van der Fits and Memelink,
2001, Yu, Li, et al., 2012)—with four methods (NJ, minimum evolution, maximum likelihood, and unweighted
pair group method with arithmetic mean [UPGAMA]).
The tree was grouped into three clades in the NJ tree (Fig.
S7A). Sm108, Sm128, Sm139, and Sm152 proteins were
clustered with all of the functional TFs in clade 1, and
Sm026 was grouped in clade 2. Sm108, Sm128, Sm139,
and Sm152 all belong to the ERF-B3 subgroup, whereas
Sm026 is a member of the DREB-A6 subgroup. The phylogenetic relationships of the other trees were similar
with the NJ tree. According to this result, we speculate
that several members of the ERF-B3 subgroup are the
main regulators of tanshinone biosynthesis. Meanwhile,
gene coexpression analysis revealed that the expression
profiles of Sm026, Sm128, and Sm152 were coexpressed
with those of the control terpenoid synthase genes
(SmCPS1 and SmDXS2) (Table S5). Overall, these results
suggested that Sm128 and Sm152 might be involved in
tanshinone biosynthesis (Fig. 4).
Finally, to identify the cis-elements of AP2/ERF TFs,
the promoter sequences of the genes encoding enzymes
in the tanshinone biosynthesis pathway were analyzed
(Table S6). Seven cis-elements were observed for AP2/
ERF TFs. RAV1AAT was identified in the promoter
regions of all studied genes. The GCC box was the ciselements of ERF-B3 subgroup. Interestingly, many of
the pathway genes—including AACT6 (acetyl-CoA
C-acetyltransferase 6), HMGS1 (hydroxymethylglutarylCoA synthase 1), HMGR1 (hydroxymethylglutarylCoA reductase), HMGR2 (3-hydroxy-3-methyl glutaryl
coenzyme A reductase 2), PMK (5-phosphomevalonate
kinase), IPI1 (isopentenyl diphosphate isomerase 1),
GGPPS5 (geranylgeranyl diphosphate synthase 5), CPS1,
KSL1 (kaurene synthase-like 1), KSL5 (kaurene synthase-like 5), KSL7 (kaurene synthase-like 7) and KSL8
(kaurene synthase-like 8)—all had GCC box sites in their
promoter regions. This finding indicated that the expression of these genes might be regulated by AP2/ERF TFs.
We also performed synteny analysis by comparing
the sequences of candidate genes (Sm008, Sm128, Sm152,
and Sm166) with the A. thaliana genome. Only one syntenic block was identified (Fig. S8). Scaffold 766 showed
a consistent relation with chromosome 4 of A. thaliana. Compared with A. thaliana, more gene insertions
occurred in scaffold 766. No tanshinone or phenolic acid
biosynthetic genes were found in scaffold 766. Moreover,
although AT4G18450 was the best-hit gene of Sm152 in
A. thaliana, AT4G18450 has not yet been functionally
analyzed. Therefore, this result provides insight for the
characterization of gene functions.
Identification of Candidate SmAP2 Genes
Related to Phenolic Acid Biosynthesis
We used the RNA-Seq data to screen 10 genes (Sm008,
Sm038, Sm042, Sm068, Sm098, Sm121, Sm136, Sm163,
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Sm165, and Sm166) for their potential involvement in the
regulation of phenolic acid biosynthesis. The qRT-PCR
results further revealed that the expression of Sm008,
Sm038, Sm042, Sm098, Sm121 and Sm166 was highest in
the phloem and xylem and lowest in the periderm, and
their expression profiles were consistent with those of the
positive control SmRAS1 (Fig. S6).
Phylogenetic tree and gene co-expression analyses
were also performed to narrow the candidate genes. The
NJ tree (Fig. S7 A) showed that the candidate TFs were
distributed in all three clades. Sm008 and Sm166 were
closely related to the functional TFs in clade 1. The other
four TFs were slightly farther from the secondary metabolism-related TFs in clades 2 and 3. The genes predicted
to be involved in phenolic acid biosynthesis were from
different subgroups (Sm008 from ERF-B4; Sm038 and
Sm098 from DREB-A1; Sm042 from DREB-A4; Sm121
from DREB-A6; and Sm166 from ERF-B1). Only Sm008
and Sm166 were from the ERF subfamily. Meanwhile,
gene co-expression analysis revealed that Sm008 and
Sm166 were co-expressed with the control rosmarinic
acid synthase gene (SmRAS) (Table S5). These data therefore suggested that Sm008 and Sm166 might be involved
in phenolic acid biosynthesis (Fig. 4).
Promoter analysis of genes encoding enzymes in the
phenolic acid biosynthesis pathway was also performed
using online PLACE software (Table S7). In contrast
to the tanshinone biosynthesis genes, the candidate
genes related to phenolic acid biosynthesis possessed
far fewer cis-elements for AP2/ERF TFs. Similar to the
genes involved in tanshinone biosynthesis, most of the
genes in phenolic acid biosynthesis studied possessed a
RAV1AAT site. The GCC-box site was detected in the
promoter regions of 4CL3 (4-coumarate:CoA ligase 3),
4CL-like2 (4-coumarate:CoA ligase–like 2), 4CL-like4
(4-coumarate:CoA ligase–like 4), TAT1 (tyrosine aminotransferase 1), HPPR1 (4-hydroxyphenylpyruvate reductase 1), RAS1, HCT1 (hydroxycinnamoyltransferase 1),
and CYP98A78, all of which are involved in phenolic acid
biosynthesis (Huang, et al., 2008a; Huang, et al., 2008b;
Xiao et al., 2011; Di et al., 2013; Wang et al., 2014). Thus,
AP2/ERF TFs might regulate these genes to affect phenolic acid accumulation via these genes.
DISCUSSION
We comprehensively surveyed the phylogeny, gene
structure, conserved motifs, and expression profiles
of the AP2/ERF TF family in S. miltiorrhiza, a highly
economical and medicinally valuable plant, to obtain
further information for AP2/ERF TFs. In total, 170
AP2/ERF genes were identified from the S. miltiorrhiza
genome database (538 Mb). Compared with the numbers of AP2/ERF genes in other plants—A. thaliana (147
genes/genome size: 125 Mb; Sakuma et al., 2002), rice
(164 genes/genome size: 466 Mb; Nakano et al., 2006),
soybean (148 genes/genome size: 1115 Mb; Zhang et
al., 2008), V. vinifera (149 genes/genome size: 487 Mb;
Zhuang et al., 2009; Licausi et al., 2010), Chinese cabbage
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(291 genes/genome size: 485 Mb; Song et al., 2013)—the
number of AP2/ERF TFs in S. miltiorrhiza is similar
(with the exception of Chinese cabbage), indicating that
the number of AP2/ERF TFs is conserved. In addition,
this result shows that the number of AP2/ERF genes is
not related to the genome size.
A comparison of the organization of the AP2/ERF
gene family of S. miltiorrhiza with that of other species
clearly showed that DREB and ERF are the prominent
subfamilies. Chinese cabbage has 291 AP2/ERF genes,
whereas the AP2, RAV, and Soloist subfamilies have only
49, 14, and 1 members, respectively, a distribution similar
to that for A. thaliana. This finding supports the evolution of AP2/ERF TFs because the members of these three
subfamilies have more complex gene structures, such
as greater numbers of introns and additional domain
insertions, and this complexity might have impaired the
homing processes, leading to a lower duplication rate
(Magnani et al., 2004). In addition, the phylogenetic tree
comparing AP2 domain sequences between S. miltiorrhiza and V. vinifera further confirmed the classification
of the DREB and ERF subgroups. As expected, the distribution of DREB/ERF members is similar within the two
plants. These two species have no A3 subgroup, whereas
the B3 subgroup contains the largest number of members, with 37 in V. vinifera and 28 in S. miltiorrhiza. Phytohormones such as jasmonate, salicylate, and ethylene
effectively increase the contents of secondary metabolites
(Bennett and Wallsgrove, 1994; Qian et al., 2006; Kai et
al., 2012; Namdeo, 2007). Some ERF-B3 members have
been reported to integrate jasmonate, alicylate, and ethylene hormonal signal-transduction pathways (Brown et
al., 2003; Lorenzo et al., 2003; Van der Does et al., 2013).
Therefore, considering the 18 members in A. thaliana, we
predict that the B3 members in V. vinifera (37 genes) and
S. miltiorrhiza (28 genes) may be involved in the diversity
of secondary metabolites. Genome-wide identification
will provide a more comprehensive understanding of the
classification, gene structures, and phylogenetic relationships of the AP2/ERF TF family, thus further elucidating
the relationship between structure and gene function.
AP2/ERF TFs can be divided into transcriptional
activator and repressor proteins. These proteins contain several conserved motifs; however, most of them
have not yet been studied. We detected one of the most
studied repression motif (ERF-associated amphiphilic
repression [EAR] motif) in the ERF-B1 (Sm011, Sm054,
Sm105, Sm122, Sm165, and Sm166), DREB-A5 (Sm051 and
Sm052) and AP2 (Sm057, Sm081, and Sm106) subfamilies.
The EAR motif can effectively suppress the activation of
transcription by interacting with other corepressors (Ohta
et al., 2001). Another motif named BRD (B3 repression
domain), which has an RLFGV sequence, was found in
two of RAV members (Sm020 and Sm133); the TFs that
contain the BRD motif also exhibited repressive activity
(Ikeda and Ohme-Takagi, 2009). Moreover, the strong
activation motif EDLL was found in 13 members (Sm001,
Sm013, Sm014, Sm021, Sm031, Sm074, Sm083, Sm115,
the pl ant genome
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ju ly 2016
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vol . 9, no . 2
Sm139, Sm152, Sm156, Sm159, and Sm164) of the ERF-B3
subgroup. This motif, which has been used to alter flowering time in Arabidopsis, was considered a potential tool
for targeted gene activation in a study of various developmental pathways (Tiwari et al., 2012). In addition, some of
the DREB-A1 (Sm002, Sm003, Sm098, Sm111, and Sm129)
and A4 (Sm086, Sm095, and Sm123) members contain
a conserved LWSY motif in the C-terminal region; this
motif was reported in OsDREB1A/B/C and in AtCBF3/
DREB1A, which were induced by drought, high-salt, and
cold conditions (Dubouzet et al., 2003).
In metabolic-engineering research, a core TF can
activate several key enzymes of a metabolic pathway,
leading to a large increase in the production of the
desired compound (van der Fits and Memelink, 2001;
Yu et al., 2012). Moreover, genetic manipulation of TF
regulation is much easier than more transformations or
co-transformation of several key enzymes. Thus, one of
the primary objectives of this study was to predict the
candidate AP2/ERF TFs that may regulate the biosynthesis of tanshinones and phenolic acids, which are medicinal compounds that have been commonly used to treat
cardiovascular diseases for a thousand years.
Secondary metabolites usually accumulate in a
tissue-specific manner. For example, the biosynthesis
and accumulation of artemisinin occur in glandular trichomes (GTs; Olsson et al., 2009). Gossypol accumulates
primarily in pigment glands in cotton (Xu et al., 2004).
The root of S. miltiorrhiza is used as a medicine and is the
major site for tanshinone and phenolic acid accumulation.
Additionally, most of the genes encoding the enzymes in
this biosynthetic pathway are also highly expressed at the
accumulation site. These genes include ADS (amorpha4,11-diene synthase), CYP71AV1, DBR2 (double bond
reductase 2), and ALDH1 (aldehyde dehydrogenase 1)
in the GTs of A. annua (Olsson et al., 2009) as well as
SmDXS2 and SmCPS1 in the root of S. miltiorrhiza (Ma et
al., 2012). The spatiotemporal transcriptional regulation
of metabolic pathway genes is tightly controlled at different levels involving many regulatory TFs. Previous studies
have shown that the expression profiles of TFs are usually
consistent with the production of secondary metabolites
in different tissues and are similar to the expression profiles of the enzyme genes. In A. annua, both AaERF1 and
AaERF2 are highly expressed in inflorescences, similar
to ADS and CYP71AV1 (Yu et al., 2012). AaORA from
A. annua is a trichome-specific transcription factor, and
AaORA exhibits similar expression patterns to those of
ADS, CYP71AV1, and DBR2 in different tissues (Lu et al.,
2013). Using this result as a screening principle, we used
RNA-seq data to select 11 and 10 genes that potentially
regulate the biosynthesis of tanshinones and phenolic
acids, respectively. The qRT-PCR method was also used to
confirm the expression patterns of these genes. The results
indicate that five and six genes that potentially regulate the
biosynthesis of tanshinones and phenolic acids, respectively, were expressed similarly to pathway genes among
different tissues. Finally, combined with phylogenetic tree
and gene coexpression analyses, two genes (Sm128 and
Sm152) and two genes (Sm008 and Sm166) were predicted
to be involved in secondary metabolism in S. miltiorrhiza.
All of the following AP2/ERF TFs involved in the
regulation of secondary metabolism studied to date belong
to the ERF-B3 subgroup, including ORCA2 and ORCA3
from C. roseus (Menke et al., 1999; van der Fits and Memelink, 2001); AaORC, AaERF1, and AaERF2 from A. annua
( Yu et al., 2012; Lu et al., 2013); and NIC-2 locus ERF TFs
in N. tabacum (Shoji et al., 2010). Two candidate genes
(Sm128 and Sm152) involved in the biosynthesis of tanshinones belong to the ERF-B3 subgroup, revealing the significance of their potential functions. Importantly, other
ERF subgroups are likely to regulate the biosynthesis of
phenolic acid. No studies thus far have examined the roles
of AP2/ERF TFs in regulating phenolic acid biosynthesis.
Moreover, our study revealed that the Sm008 and Sm166
candidate genes were coexpressed with the key enzyme
gene (SmRAS) with Pearson correlation value of 0.948 and
0.899, respectively. In addition, the promoter sequences of
most genes in phenolic acid metabolic pathways possess
the cis-elements of AP2/ERF TFs. Therefore, the functions
of these genes are worth studying.
Supplemental Information Available
Supplemental information is included with this article.
Figure S1. AP2/ERF transcription factor distribution
in S. miltiorrhiza.
Figure S2. Comparison of the AP2/ERF domain
sequences of the AP2 (A), ERF (B), DREB (C), RAV (D),
and Soloist (E) subfamilies proteins between A. thaliana
and S. miltiorrhiza. The blue background indicates the
conservation of amino acid residues (100%). The pink
background indicates the conservation of amino acid
residues (75%).
Figure S3. Phylogenetic tree constructed using the
AP2/ERF domain sequences of the DREB and ERF subfamilies of S. miltiorrhiza and V. vinifera.
Figure S4. The features of the gene structures and
the distribution of the conserved motifs of the AP2 (A),
DREB (B), RAV (C), and Soloist (D) subfamilies of S.
miltiorrhiza. (a) Phylogenetic relationships among each
subgroups. (b) The distribution of the conserved motifs
of each protein. (c) Exon/intron structures of the AP2/
ERF genes of S. miltiorrhiza.
Figure S5. The expression profiles of S. miltiorrhiza
AP2/ERF genes in the periderm, phloem and xylem. A,
B, C, and D indicate the expression patterns of the AP2,
ERF, DREB, and RAV-Soloist subfamilies, respectively.
The color bar at the top represents the expression values. The genes in white were not expressed in these four
organs and the FPKM (fragments per kilobase of exon
model per million mapped reads) values were zero.
Figure S6. Expression patterns of selected AP2/ERF
genes related to tanshinone (A) (five genes) and phenolic
acid (B) (six genes) biosynthesis. Relative expression of the
selected AP2/ERF genes in R (root), S (stem), L (leaf), F
(flower), R1 (periderm), R2 (phloem), and R3 (xylem) was
ji et al .: genome - wi de analysis of ap 2 in salvia mi ltiorrhiz a 9
of
11
examined by qRT-PCR. The characters on the x axis indicate different organs and tissues. The y axis shows the fold
changes in gene expression. The S. miltiorrhiza actin gene
was used as an internal reference. SmDXS2, SmCPS1, and
SmRAS1 were tested as positive controls. We performed
one-way ANOVA using IBM SPSS 20 software. Asterisks
representing significant differences from this comparison.
P < 0.01 was considered highly significant.
Figure S7. Phylogenetic tree constructed using the AP2
domain sequences of the 11 candidate TFs of S. miltiorrhiza
and other medicinal plant AP2s associated with secondary metabolism. The phylogenetic tree was constructed
using the neighbor-Joining (A), minimum evolution (B),
UPGMA (C), and maximum likelihood (D) method.
Figure S8. Synteny relationships between S. miltiorrhiza Sm152 and A. thaliana AT4G18450 loci. The colored
boxes represent the different types of genes.
Table S1. Primer sequences of the candidate genes
related to the biosynthesis of tanshinones and phenolic
acids used for qRT-PCR analyses.
Table S2. Complete list of the AP2/ERF transcription
factors identified in the S. miltiorrhiza genome.
Table S3. Conserved motifs identified from the AP2/
ERF genes in S. miltiorrhiza.
Table S4. The average FPKM values of 170 AP2/ERF
genes in different organs and tissues.
Table S5. Coexpression analysis of candidate genes
and pathway genes using Pearson’s correlation test.
Table S6. Cis-acting regulatory elements targeted by
AP2/ERF TFs in the promoters of the key enzyme genes
of the tanshinone metabolic pathway.
Table S7. Cis-acting regulatory elements targeted by
AP2/ERF TFs in the promoters of the key enzyme genes
of the phenolic acid metabolic pathway.
Acknowledgments
This work was supported by the National Natural Science Foundation (grant
nos. 81373916 and 81573398) and the Program for Innovative Research Team
at the Institute of Medicinal Plant Development (grant no. IT1304).
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