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Genome-wide analysis of the
ERF Family in Stephania japonica
provides insights into
the regulatory role in
Cepharanthine biosynthesis
Hanting Yang
1,2
†
, Baimei Liu
1,2
†
, Haiyan Ding
1
, Zhaoyu Liu
2,3
,
Xiaodong Li
4
, Tianxing He
1
,YaWu
1
, Yuxuan Zhang
1
,
Can Wang
1,2
*, Liang Leng
1,2
*, Shilin Chen
1,2
*and Chi Song
1,2
*
1
School of Pharmacy/School of Modern Chinese Medicine Industry, Chengdu University of Traditional
Chinese Medicine, Chengdu, China,
2
Institute of Herbgenomics, Chengdu University of Traditional
Chinese Medicine, Chengdu, China,
3
School of Chinese Materia Medica, Tianjin University of
Traditional Chinese Medicine, Tianjin, China,
4
Wuhan Botanical Garden, Chinese Academy of
Sciences, Wuhan, China
Introduction: Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid (bisBIA)
extracted from Stephania japonica, has received significant attention for its
anti-coronavirus properties. While ethylene response factors (ERFs) have been
reported to regulate the biosynthesis of various alkaloids, their role in regulating
CEP biosynthesis remains unexplored.
Methods: Genome-wide analysis of the ERF genes was performed with
bioinformatics technology, and the expression patterns of different tissues,
were analyzed by transcriptome sequencing analysis and real-time quantitative
PCR verification. The nuclear-localized ERF gene cluster was shown to directly
bind to the promoters of several CEP-associated genes, as demonstrated by
yeast one-hybrid assays and subcellular localization assays.
Results: In this work, 59 SjERF genes were identified in the S. japonica genome
and further categorized into ten subfamilies. Notably, a SjERF gene cluster
containing three SjERF genes was found on chromosome 2. Yeast one-hybrid
assays confirmed that the SjERF gene cluster can directly bind to the promoters
of several CEP-associated genes, suggesting their crucial role in CEP
metabolism. The SjERFs cluster-YFP fusion proteins were observed exclusively
in the nuclei of Nicotiana benthamiana leaves. Tissue expression profiling
revealed that 13 SjERFs exhibit high expression levels in the root, and the qRT-
PCR results of six SjERFs were consistent with the RNA-Seq data. Furthermore, a
co-expression network analysis demonstrated that 24 SjERFs were highly
positively correlated with the contents of various alkaloids and expression
levels of CEP biosynthetic genes.
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Weizhen Liu,
Wuhan University of Technology, China
REVIEWED BY
Pawan Kumar,
Agricultural Research Organization
(ARO), Israel
Xiaoxu Li,
Chinese Academy of Agricultural
Sciences (CAAS), China
*CORRESPONDENCE
Can Wang
wangcan@cdutcm.edu.cn
Liang Leng
lling@cdutcm.edu.cn
Shilin Chen
slchen@cdutcm.edu.cn
Chi Song
songchi@cdutcm.edu.cn
†
These authors have contributed equally to
this work
RECEIVED 15 May 2024
ACCEPTED 15 August 2024
PUBLISHED 04 September 2024
CITATION
Yang H, Liu B, Ding H, Liu Z, Li X, He T, Wu Y,
Zhang Y, Wang C, Leng L, Chen S and Song C
(2024) Genome-wide analysis of the ERF
Family in Stephania japonica provides
insights into the regulatory role in
Cepharanthine biosynthesis.
Front. Plant Sci. 15:1433015.
doi: 10.3389/fpls.2024.1433015
COPYRIGHT
©2024Yang,Liu,Ding,Liu,Li,He,Wu,Zhang,
Wang, Leng, Chen and Song. This is an open-
access article distributed under the terms o f
the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction
in other forums is permitted, provided the
original author(s) and the copyright owner(s)
are credited and that the original publication
in this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 04 September 2024
DOI 10.3389/fpls.2024.1433015
Conclusion: This study provides the first systematic identification and analysis of
ERF transcription factors in the S.japonica genome, laying the foundation for the
future functional research of SjERFs transcription factors.
KEYWORDS
ERF,Stephania japonica, Cepharanthine biosynthesis, expression patterns, genome-
wide analysis
1 Introduction
The COVID-19 outbreak in 2019 had a severe global impact,
prompting scientists worldwide to collaborate in the search for
effective drugs (Li et al., 2022;Yang et al., 2024a). Cepharanthine
(CEP) has demonstrated the ability to inhibit the entry of SARS-
CoV-2 into cells by blocking the virus’s attachment to its intended
target cells (Kumar et al., 2022). This characteristic makes CEP a
promising therapeutic agent for potential anti-COVID-19
treatments (Kumar et al., 2022;Fan et al., 2020). CEP, a bisBIA
isolated from Stephania japonica, with the biological activities of
antioxidant (Chen et al., 2019), antitumor (Zhang et al., 2021), and
immunomodulatory (Xu et al., 2021). CEP predominantly
accumulates in the roots of S. japonica, followed by the leaves
and stems (Leng et al., 2024). S. japonica (Thunb.) Miers, a tangled
deciduous woody vine belonging to the Menispermaceae family and
Stephania genus (Al-Amin et al., 2022), is commonly used in
traditional Chinese folk medicine for its heat-clearing,
detoxifying, and “wind and blockage”dispelling properties in the
human body (Xiao et al., 2019). Given the increasing clinical
demand for CEP, it is crucial to investigate its biosynthesis and
transcriptional regulation.
The biosynthesis of CEP primarily initiates with dopamine and 4-
hydroxyphenyl acetaldehyde catalyzed by norclaurane synthesis
(NCS) (Minami et al., 2007), norclaurane 6-O-methyltransferase
(6OMT) (Li et al., 2020), coclaurine N-methyltransferase (CNMT)
(Zhao et al., 2020), and undergoes multi-step reaction catalyzed by
OMT/CYP80A (Stadler et al., 1988;Carina and Till, 2019). The oxidase
CYP80A1 selectively couples two N-methylcocoyl units in their
benzylic portion, forming the simplest bisBIA (Kraus and Kutchan,
1995). The biosynthesis of guattegaumerine and berbamunine of
bisBIA s has been elucidated (Payne et al., 2021). In S. japonica,
SjNCS2 and SjNCS4 possessed NCS functionality and exhibited
superior enzymatic activities compared with the Coptis chinensis
NCS (Leng et al., 2024). However, the downstream biosynthetic
pathways of CEP remain unclear (Supplementary Figure S1).
ERF transcription factors are significant regulators in various
plant biological processes, including alkaloid biosynthesis (Feng
et al., 2020;Yamada and Sato, 2021). For instance, clustered ORCA
transcription factors (ORCA2-6) regulate the expression of different
monoterpene indole alkaloid biosynthetic genes in Catharanthus
roseus (Paul et al., 2020;Singh et al., 2020). In Nicotiana
benthamiana,NtERF189 acts as a master regulator of nicotine
biosynthesis by recognizing GCC-box-like elements in the
promoter of nicotine biosynthetic genes (Shoji et al., 2010;Shoji
and Hashimoto, 2012). OpERF2 positively regulates the anti-cancer
camptothecin biosynthesis in Ophiorrhiza pumila (Udomsom et al.,
2016). In Eschscholzia californica, a luciferase reporter assay
indicated that four Group IX AP2/ERF TFs, known as EcERF2,
EcERF3,EcERF4, and EcERF12, can trans-activate Ec6OMT and
EcCYP719A5, which are involved in BIA biosynthesis (Yamada
et al., 2020). Transiently overexpressing PhERF1 in petunia leaves
has an impact on the production of petuniolides and petuniaserones
(Shoji et al., 2023). Overexpression of ScAPD1-like significantly
increased the metabolites of the phenylpropanoid pathway by
directly regulating the abundance of ScPAL and ScC4H transcripts
(Li et al., 2023). The ERF transcription factor WAX INDUCER1
(WIN1) promotes the accumulation of total polyphenols in
Nicotiana tabacum, including chlorogenic acid (He et al., 2024).
However, a study on the ERF family in S. japonica that regulates the
biosynthesis of bisBIA has yet to be reported.
An increasing number of medicinal plant genomes have been
published, including Artemisia argyi,Mentha suaveolens, and C.
roseus, which will provide a foundation for the identification of ERF
families and functional genomics research (Chen et al., 2023;Yang
et al., 2024b;Sun et al., 2023;Pei et al., 2024). ERF protein
identification and characterization have been studied in various
plant species, including Arabidopsis thaliana (Nakano et al., 2006),
barley (Taketa et al., 2008), Fagopyum Tataricum (Liu et al., 2019),
grape (Zhuang et al., 2009;Zhu et al., 2019), apple (Girardi et al.,
2013), and ginger (Xing et al., 2021). The number of ERF TFs family
in many plants are as follows: 136 (Oryza sativa), 122 (A. thaliana),
96 (Citrus junos), 92 (Camptotheca acuminata), 80 (Vitis vinifera),
and 60 (E. californica). Genome-wide identification of ERF
transcription factor and its significance in CEP biosynthesis have
not been elucidated in Stephania plants. This study proved the
systematic identification and analysis of 59 SjERFs in the S. japonica
genome using a set of bioinformatics tools. Meanwhile, tissue
expression profiling and co-expression analysis of SjERF,CEP
biosynthetic genes, and BIAs metabolites were also conducted.
Yeastone-hybridassaysindicatedthattheSjERFs cluster
recognizes GCC boxes in the promoters of several CEP-associated
Yang et al. 10.3389/fpls.2024.1433015
Frontiers in Plant Science frontiersin.org02
genes. This work provides valuable insight into the roles of ERF
transcription factors in CEP biosynthesis and enhances our
understanding of the ERF gene family in plants.
2 Materials and methods
2.1 Plant materials
The S. japonica plants were cultivated and harvested in Wuhan,
Hubei Province, China. Different tissues of S. japonica including
stems, leaves, roots, and shoots, were collected for transcriptome
sequencing and quantitative real-time polymerase chain reaction
(qRT-PCR) experiments. Three biological replicates were
conducted for each experiment.
2.2 Identification of SjERF genes in the
S. japonica genome
Our research group has acquired the genome data of S.
japonica., and has been archived under the China National
GeneBank DataBase (CNGBdb) accession number CNP0003595
(https://db.cngb.org/search/?q=CNP0003595)(Leng et al., 2024).
The AtERFs protein sequences of A. thaliana were downloaded
from the Arabidopsis Information Resource (TAIR) database
(http://www.arabidopsis.org/). The hidden Markov model (HMM,
PF00847) was used to search for ERF candidate genes in the S.
japonica genome, with a threshold of 0.01. Furthermore, to ensure
the comprehensive identification of SjERF genes, 121 AtERF
proteins were used to BLAST the S. japonica protein database for
ERF-containing sequences (Supplementary Table S1), minimizing
the risk of missing any SjERF genes. Then, candidate proteins with
only one AP2 domain were manually screened (Sakuma et al., 2002;
Riechmann and Meyerowitz, 1998). The Molecular weight (MW)
and pI of SjERF proteins were analyzed using the Expasy website
(https://prosite.expasy.org/). Finally, the subcellular localization of
SjERFs was predicted using WoLF PSORT and CELLO online
software (Horton et al., 2007).
2.3 Classification, gene structure, and
protein motif analysis of SjERF genes in
S. japonica
To explore different biological characteristics and evolutionary
relationships of SjERF proteins in S. japonica,anunrooted
phylogenetic tree of ERFs protein sequences (59 SjERFs and 121
AtERFs) from S. japonica and A. thaliana was constructed by
MEGA11 with 1,000 bootstrap replicates (Tamura et al., 2021).
Then, an evolutionary tree was beautified and decorated using
Evolview (Zhang et al., 2012). The conserved motifs of SjERFs
protein were identified using the MEME website (parameters:
number of motifs: 10, wide: 10-50, others are default values)
(Bailey et al., 2009). Gene structure and protein motif of SjERFs
were visualized using TBtools (Chen et al., 2020).
2.4 Analysis of cis-elements, microsynteny,
and evolutionary patterns of SjERF genes
The promoter sequences of the 59 SjERFs (-2,000 to -1 bp) were
extracted using TBtools. Subsequently, cis-acting regulatory elements
of SjERFs gene promoters have been predicted and identified by
PlantCARE (Lescot et al., 2002). The chromosomal positions of
SjERF genes were retrieved from the S. japonica genome database
and graphically represented using TBtools software. The duplication
events of the SjERFs were analyzed using MCScanX and BLASTP
(Wang et al., 2012). The synonymous relationship between SjERFs and
AtERFs,OsERFs,CrERFs,andNtERFs was analyzed and visualized by
TBtools software. The genome data of O. sativa,C. roseus,andN.
tabacum were retrieved from the National Center for Biotechnology
Information (NCBI: https://www.ncbi.nlm.nih.gov/), respectively.
2.5 Chromosome structure prediction and
cluster prediction
The Topologically Associated Domains (TADs) were identified
based on previous reports (Sun et al., 2020). Initially, the Hi-C read
pairs were aligned to the S. japonica genome, and contact matrixes
were generated using HiC-Pro (Servant et al., 2015). Subsequently,
the Hi-C contact matrixes were imported into HiCExplorer (Wolff
et al., 2018) and converted using the built-in function
(hicConvertFormat). Then, the contact matrixes were normalized
using hicNormalize with the KR correction method and corrected
using hicCorrectMatrix with a filter threshold of -1.5 to 5. Next, the
hicFindTADs algorithm was applied to identify TADs at various
resolutions. The specific parameters used for this analysis were a
minimum depth of 5, maximum depth of 10, step size of 2, and a
threshold for comparisons set at 0.01.
2.6 Yeast one-hybrid assays
Yeast one-hybrid assays were performed to determine whether
SjERF9-11 could bind to the GCC motif. The functional protein
sequences of CEP biosynthetic genes with known functions were
retrieved from the NCBI database, including NCS,6OMT, and
CNMT (Supplementary Table S2). The candidate genes involved in
CEP biosynthesis were predicted using BLASTP (option: e-value
1e
−10
). Subsequently, the functional proteins and candidate genes
were used to construct phylogenetic trees with 1,000 bootstrap
replicates. Additionally, the ERF binding elements in CPE
biosynthetic gene promoters were predicted using PlantCARE
software. The open reading frame (ORF) fragment of SjERF9-11
was individually cloned into the effector plasmid pB42AD.
Additionally, the triple tandem copy of the GCC motif
(GCCGCC) or the ERF binding element from CEP-biosynthetic
gene promoters was inserted into the reporter plasmid pLacZ. The
effector and reporter plasmids were jointly transformed into the
yeast strain EGY48 and grown on SD/-Ura/-Trp medium.
Subsequently, the co-transformed cells were assayed on SD/-Ura/-
Yang et al. 10.3389/fpls.2024.1433015
Frontiers in Plant Science frontiersin.org03
Trp medium containing 5-bromo-4-chloro-3-indolyl-b-D-
galactopyranoside (X-gal) for 24 hours, as previously described
(Wang C. et al., 2022). Empty plasmids (pB42AD and pLacZ) were
used as a negative control for the transformation. All primers
utilized in this study are provided in Supplementary Table S3.
2.7 Subcellular localization
To analyze the subcellular localization of three SjERFs, the
SjERF9-11 ORF fragments were amplified and individually
integrated into the modified plant expression vector pHB-YFP.
The plasmids pHB-SjERFs-YFP and the empty vector pHB-YFP
(serving as the negative control) were introduced into the
Agrobacterium tumefaciens strain GV3101 and transiently
infected the epidermal cells of 5-week-old N. benthamiana leaves,
as previously described (Wang C. et al., 2021;Hao et al., 2023). YFP
signals were analyzed 48 h post-infection using an LSM880 confocal
laser microscope (Carl Zeiss, Germany). Nuclei were stained with,
4’6-diamidino-2-phenylindole (DAPI, Sigma, Code No. D9542).
Three biological replicates were performed as reported previously,
to ensure the reliability of the results.
2.8 RNA-seq and qRT-PCR
For RNA-seq analysis, qualified RNA samples underwent
testing for database establishment. The quality of the constructed
library was assessed using an Agilent 2100 Bioanalyzer, while
sequencing was performed using DNBSEQ technology. All raw
sequencing data have been deposited under the National Center for
Biotechnology Information (NCBI) GenBank accession number
PRJNA888087. The expression pattern of SjERFsindifferent
tissues was analyzed by TBtools according to the FPKM values.
Total RNA was extracted from the roots, stems, leaves, and shoots
of S. japonica using a plant total RNA Extraction Kit (Foregene
Biotech, Chengdu, China, Code No. RE-05011). Subsequently,
reverse transcription was carried out according to the instructions
provided with the gDNA Eraser reagent Kit (Foregene Biotech,
Chengdu, China, Code No. RT-01032) for qRT-PCR analysis. The
qRT-PCR was carried out according to previous reports, and three
biological replicates were conducted for each experiment. For qRT-
PCR normalization, SjGAPDH, a housekeeping gene in S. japonica,
was employed as an internal control of all samples (Yang et al.,
2023;Jain et al., 2018;Barber et al., 2005). The specific primers used
for the analysis are detailed in Supplementary Table S3. The relative
expression levels of SjERFs across various tissues were determined
using the 2
−DDCt
method.
2.9 Co-expression network of SjERFs
involved in CEP biosynthesis pathway
46 SjERFs and 9 CEP biosynthetic genes all exhibiting FPKM
values exceeding 1, underwent co-expression analysis using
Pearson’s correlation test. We employed untargeted metabolomics
TABLE 1 Sequence characteristics of 59 SjERFs.
ID Gene
name Type Chr Start end Strand No.
of Exon
CDS
length
Mw
(Da) pI Loc
SjapChr1G00001760.1 SjERF1 I chr1 2711408 2714932 + 1 1194 43737.41 5.24 Nuc
SjapChr1G00003160.1 SjERF2 VIII chr1 4557505 4557987 + 1 486 16797.81 9.51 Nuc
SjapChr1G00003930.1 SjERF3 IX chr1 5417393 5417956 + 1 567 20593.55 6.67 Nuc
SjapChr1G00008120.1 SjERF4 V chr1 11728956 11729894 –1 942 34746.61 5.65 Nuc
SjapChr1G00012460.1 SjERF5 III chr1 19185052 19189635 + 2 669 24567.25 5 Nuc
SjapChr1G00015200.1 SjERF6 V chr1 23733363 23736700 –2 1077 39871.19 5.22 Nuc
SjapChr1G00029910.1 SjERF7 VIII chr1 69410464 69416430 –1 741 27653.31 9 Nuc
SjapChr2G00042580.1 SjERF8 III chr2 4844300 4845016 + 1 720 25621.04 5.14 Nuc
SjapChr2G00045100.1 SjERF9 IX chr2 9308650 9309432 –1 786 28946.91 4.66 Nuc
SjapChr2G00045110.1 SjERF10 IX chr2 9405916 9406914 –1 999 36667.17 8.77 Nuc
SjapChr2G00045130.1 SjERF11 IX chr2 9534735 9535544 + 1 813 28876.08 9.40 Nuc
SjapChr2G00053160.1 SjERF12 I chr2 41808331 41809793 –1 1008 36626.76 8.98 Nuc
SjapChr2G00054830.1 SjERF13 III chr2 46323345 46324282 –1 510 18666.3 8.64 Nuc
SjapChr2G00059370.1 SjERF14 chr2 56313792 56319526 + 6 684 25387.47 9.16 Nuc
SjapChr2G00063270.1 SjERF15 IX chr2 63234204 63235384 –1 876 32963.44 6.13 Nuc
SjapChr2G00063280.1 SjERF16 IX chr2 63280657 63285641 –1 438 15963.47 5.74 Nuc
(Continued)
Yang et al. 10.3389/fpls.2024.1433015
Frontiers in Plant Science frontiersin.org04
TABLE 1 Continued
ID Gene
name Type Chr Start end Strand No.
of Exon
CDS
length
Mw
(Da) pI Loc
SjapChr2G00064000.1 SjERF17 VIII chr2 64448707 64449231 + 1 528 18808.37 9.88 Nuc
SjapChr2G00067800.1 SjERF18 VIII chr2 70373807 70374925 + 1 1122 41653.22 5.75 Nuc
SjapChr3G00074020.1 SjERF19 VII chr3 4479441 4480956 + 2 720 26910.39 8.92 Nuc
SjapChr3G00074030.1 SjERF20 VII chr3 4505717 4508080 + 2 774 29030.07 5.08 Nuc
SjapChr3G00075730.1 SjERF21 II chr3 6760242 6765099 + 1 648 23872.71 5.55 Nuc
SjapChr3G00085580.1 SjERF22 IX chr3 39428058 39428629 + 2 465 17471.53 6.11 Nuc
SjapChr3G00085600.1 SjERF23 IX chr3 39473034 39473534 –1 504 18653.14 5.63 Nuc
SjapChr3G00085660.1 SjERF24 VIII chr3 39611535 39611924 –1 393 14472.99 5.93 Nuc
SjapChr3G00085680.1 SjERF25 IX chr3 39818424 39819128 + 1 708 26528.49 5.65 Nuc
SjapChr3G00087630.1 SjERF26 I chr3 44029745 44030869 + 1 1128 41945.4 8.99 Nuc
SjapChr4G00102600.1 SjERF27 IV chr4 7719916 7721567 –2 561 20506.74 9.94 Nuc
SjapChr4G00109960.1 SjERF28 V chr4 20749186 20749883 –2 579 21130.05 8.98 Nuc
SjapChr4G00119750.1 SjERF29 II chr4 54210503 54212473 –1 627 23255.42 4.53 Nuc
SjapChr4G00120260.1 SjERF30 VII chr4 55128464 55131323 –2 1167 42775.32 5.08 Nuc
SjapChr5G00123460.1 SjERF31 III chr5 3514506 3515081 + 1 579 20654.48 5.16 Nuc
SjapChr5G00123480.1 SjERF32 III chr5 3559387 3560297 –1 612 22578.62 6.53 Nuc
SjapChr5G00128870.1 SjERF33 chr5 12976168 12982139 –7 1131 41961.31 6.43 Nuc
SjapChr5G00130040.1 SjERF34 V chr5 16586160 16587790 + 2 1137 41024.09 5.96 Nuc
SjapChr5G00131070.1 SjERF35 IV chr5 20244188 20245033 –1 849 32297.1 6.03 Nuc
SjapChr5G00138660.1 SjERF36 VIII chr5 45874943 45875620 + 2 597 21668.48 5.49 Nuc
SjapChr5G00141910.1 SjERF37 IV chr5 50758866 50759834 –1 972 35602.09 6.78 Nuc
SjapChr5G00142510.1 SjERF38 IV chr5 51470041 51474131 –3 1020 36785.04 9 Nuc
SjapChr5G00143880.1 SjERF39 VI chr5 53358292 53359068 + 1 780 29876.48 7.71 Nuc
SjapChr6G00156070.1 SjERF40 V chr6 35351515 35352727 –2 1110 40906.17 4.82 Nuc
SjapChr6G00166910.1 SjERF41 X chr6 52402579 52406263 + 2 867 31918.89 6.52 Nuc
SjapChr7G00173600.1 SjERF42 II chr7 8526747 8528220 –1 489 18343.55 6.84 Nuc
SjapChr7G00180830.1 SjERF43 III chr7 33074754 33075338 –1 588 21210.32 4.93 Nuc
SjapChr7G00186210.1 SjERF44 X chr7 47780599 47783481 + 2 1308 47552.22 6.05 Nuc
SjapChr7G00187000.1 SjERF45 VIII chr7 48993478 48993924 –1 450 16890.64 6.37 Nuc
SjapChr7G00187590.1 SjERF46 I chr7 49815906 49817084 + 1 1182 43619.62 6.76 Nuc
SjapChr8G00192880.1 SjERF47 V chr8 4263704 4264879 –2 651 23484.28 9.07 Nuc
SjapChr8G00193970.1 SjERF48 VIII chr8 5595150 5598496 + 2 843 30641.97 9.07 Nuc
SjapChr8G00196850.1 SjERF49 II chr8 9861774 9862899 –1 639 23020.41 4.84 Nuc
SjapChr8G00207160.1 SjERF50 V chr8 44219929 44220864 –1 939 33255.74 9.6 Nuc
SjapChr8G00207340.1 SjERF51 III chr8 44607041 44608247 –1 675 24660.28 5.14 Nuc
SjapChr9G00214400.1 SjERF52 III chr9 2802755 2804156 –1 570 20702.11 5.3 Nuc
SjapChr9G00215550.1 SjERF53 II chr9 4472534 4474762 –2 630 23032.85 10 Nuc
SjapChr9G00224540.1 SjERF54 IX chr9 37203805 37204545 + 1 744 26893.47 6.59 Nuc
(Continued)
Yang et al. 10.3389/fpls.2024.1433015
Frontiers in Plant Science frontiersin.org05
to profile BIAs across various tissues of the S. japonica (Leng et al.,
2024). According to the expression patterns of SjERFs, two BIA
precursors, alongside 23 BIA-type structures in roots, stems, and
leaves of S. japonica,thepartialcorrelationcoefficient (PCC)
method was used to calculate the Pearson correlation coefficient.
The co-expression network of SjERFs, CEP biosynthetic genes, and
BIAs metabolites was exhibited using Cytoscape, with the following
parameters: absolute value of correlation coefficient > 0.9 and p-
value < 0.05 (Shannon et al., 2003). The correlations between
SjERFs, CEP biosynthetic genes, and BIAs metabolites were
displayed in cluster heatmap using TBtools software.
3 Results
3.1 Genome-wide identification of 59 SjERF
TFs in S. japonica genome
59 non-redundant SjERF genes have been identified in the S.
japonica genome using HMMER and BLAST (Table 1). All
identified ERF genes in S. japonica were named SjERF1- SjERF59
according to their chromosome distribution (Huang et al., 2020).
All SjERFs were then manually confirmed by CDD’s online software
and a Simple Modular Architecture Analysis Tool (SMART) for the
presence of a core domain (Supplementary Figure S2). The CDS
sequence length of SjERF genes was between 605 bp (SjERF21) and
1305 bp (SjERF29), encoding 202–434 amino acids (Supplementary
Table S4). The molecular weight (Mw) of SjERFs ranged from 22.58
kDa (SjERF21) to 47.55 kDa (SjERF29), with theoretical pI values
ranging from 4.53 (SjERF19) to 10.00 (SjERF37). Almost all SjERFs
were predicted to be located in the nucleus, only SjERF57 was
located in the cytoplasmic (Table 1).
3.2 Phylogenetic relationship of
SjERF genes
The unrooted phylogenetic tree of 59 SjERFs and 121 AtERFs
has been constructed to explore the evolutionary relationship. 59
SjERFs have been divided into 10 subgroups, namely, groups I to X.
A previous study has further divided the ERF family into ERF and
CBF/DREB subfamily, and the ERF subfamily always classified into
six groups (B1 to B6) (Zhang et al., 2015). In this analysis, group I to
IV belong to the DREB subfamily, and group IV to X, and VI-L
belong to the ERF subfamily, and there is no SjERF in group Xb-L.
SjERF1, 12, 26, 46, and 59 were branched into group I, SjERF21, 29,
42, 49, 53, and 56 were branched into group II, group IX was the
largest group with 11 members (SjERF3, 9, 10, 11, 15, 16, 22, 23, 25,
54, 55). As shown in Figure 1, group VI was the smallest with
SjERF39,SjERF58 belongs to group VI-L, and SjERF14 and SjERF33
don’t belong to any subfamily.
3.3 Gene structure and motif analysis of
SjERFs in S. japonica genome
To better understand the evolution and structural diversity of
the S. japonica ERF family, the MEME (Multiple Em for Motif
Elicitation) was used to analyze the conserved sequence of the 59
SjERFs protein. The basic information (width and best possible
match sequence) of the consensus sequences of these motifs are
shown in Supplementary Table S5. The frequent motifs of SjERFs
are motif1 (RVWLGTFDTAEEAARAYDEAAFKLRG), motif2
(YRGVRQRPWGKWVAEIRDP), and motif3 (SKAKLNFPEE).
The results showed that each motif contained 10-29 kinds of
amino acids, and each SjERF contained motif1. Almost all SjERFs
contain motif2, only SjERF14 and SjERF33 don’t belong to any
subfamily that does not contain motif2, while they only contain one
conserved motif (motif1), and SjERF33 had two conserved motifs
(motif1, motif3). SjERF44,SjERF18,SjERF37,SjERF31,SjERF59,
and SjERF7 had six conserved motifs. 59 SjERFs contained ERF
conservative domain (Figure 2B;Supplementary Figure S3).
Additionally, these different motif patterns show their degree of
deviation among different groups. For example, motif 5 is the
representative of group IX. Motif 7 is only found in group III.
Motif 6 is unique to group II and VII (Figures 2A,B).
In this study, gene structures of 19 SjERFs(SjERF5, 6, 14, 19, 20,
22, 27, 28, 30, 33, 34, 36, 38, 40, 41, 44, 47, 48, 53) have one intron,
and SjERF38,SjERF33, and SjERF14 contained two or more introns.
The other 40 SjERFs contain only one exon and no intron,
accounting for 67.8% of the total SjERFsinS. japonica.Most
SjERF genes contained only one exon in group I, II, III, and IX,
except for SjERF53,SjERF5, and SjERF22. The unnamed subfamily
genes (SjERF14,SjERF33) have 6 and 7 exons (Figure 2C).
TABLE 1 Continued
ID Gene
name Type Chr Start end Strand No.
of Exon
CDS
length
Mw
(Da) pI Loc
SjapChr9G00224550.1 SjERF55 IX chr9 37238076 37238624 –1 552 20025.39 6.41 Nuc
SjapChr9G00228980.1 SjERF56 II chr9 45336446 45336937 + 1 513 19254.35 8.55 Nuc
SjapChr10G00242090.1 SjERF57 VII chr10 32748182 32748913 –1 735 28138.51 5.09 Nuc
SjapChr10G00244770.1 SjERF58 VI-L chr10 39579633 39581894 + 1 1014 37430.77 5.44 Cyt
SjapChr11G00256330.1 SjERF59 I chr11 8273391 8274458 + 1 1071 40157.51 7.8 Nuc
Loc, Subcellular location; Nuc, Nucleus; Cyt, Cytoplasmic.
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3.4 Analysis of cis-acting elements in the
SjERF genes promoter
The study identified various cis-acting elements located in the
promoter regions of SjERFs, with the majority participating in
hormone responses, and abiotic and biotic stress. In plant growth
and development, 31 CAT-boxes were implicated in meristem
expression across 26 SjERFs promoter regions, while 7 A-boxes
participated in meristem expression in 7 SjERFs promoter regions
(Figure 3;Supplementary Table S6). Furthermore, 18 GCN4_motif,
4 HD-Zip, 36 O
2
-site, 16 circadian control elements, and 7 seed-
specific regulation elements were identified in the promoter regions
of SjERFs (Supplementary Figure S4). In hormone responses,
various cis-acting regulatory elements were identified, including
164 ABRE, 33 TGA-element, 7 AuxRR-core, 4 TGA-box, 13 GARE-
motif, 10 TATC-box, 22 P-box, 123 CGTCA and TGACG-motif.
However, the abiotic and biotic stress cis-acting elements were not
found in the promoter regions of SjERF2, 7, 32, and 50.
3.5 Chromosome distribution and synteny
analysis of SjERFs
Chromosome localization analysis found that 59 SjERFs were
disproportionately distributed on eleven S. japonica chromosomes
(Figure 4A). Seven SjERFs were distributed on Chr1 and Chr3,
eleven SjERFs on Chr2, four SjERFs distributed on Chr4, nine
SjERFs distributed on Chr5, two SjERFs distributed on Chr6 and
Chr10, five SjERFs distributed on Chr7, Chr8 and Chr9, and only
one SjERF distributed on Chr11. Interestingly, three SjERF genes
containing SjERF9,SjERF10, and SjERF11 were distributed on S.
japonica chromosome 2 (9.30 - 9.54 Mb) and formed an ERF gene
cluster. Similar results have also been found in C. roseus and N.
tabacum, such as the ORCA gene cluster and NICOTINE2 (NIC2)
ERF cluster (Yuan, 2020;Shoji et al., 2010;Shoji and Yuan, 2021).
The phylogenetic tree showed that SjERF9,SjERF10, and SjERF11
and functional ERF cluster were converging into one branch, and
belonging to the IX subfamily (Figure 4B). Additionally, three
FIGURE 1
Phylogenetic tree of 59 SjERFs and 121 AtERFs. The ERF protein sequences of S. japonica and A. thaliana were used to construct the phylogenetic
tree using the Neighbor-Joining (NJ) method, with 1,000 bootstrap replicates.
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SjERFs and four other genes were located in the same topologically
associating domains (TADs) region (Figure 4C). Overall, the SjERF
gene cluster found in S. japonica genome may play an important
role in the biosynthesis of secondary metabolism.
Through collinear analysis, the potential relationship and gene
duplication type between SjERF genes in the S. japonica genome
were explored (Figure 4A). A total of seven SjERF genes were found
in three segmental duplication events, such as Chr1(SjERF1)/Chr11
(SjERF59), Chr1(SjERF5)/Chr5(SjERF31), Chr1(SjERF6)/Chr5
(SjERF38), Chr7(SjERF42)/Chr9(SjERF56), Chr4(SjERF29)/Chr8
(SjERF49). To further infer the evolutionary mechanism of the
ERF family in S. japonica, we constructed a synteny diagram of S.
japonica with C. roseus,N. tabacum,A. thaliana, and O. sativa
(Supplementary Figure S5). Between S. japonica and A. thaliana,O.
sativa,C. roseus, and N. tabacum, 56, 46, 50, and 32 syntenic SjERF
gene pairs were identified, respectively (Supplementary Table S7).
Some SjERF genes had multiple orthologous gene pairs (one SjERF
associated with multiple AtERFs). For instance, three synteny events
occurred in three SjERFs, such as SjERF9,SjERF30, and SjERF41.
Interestingly, the SjERF1,SjERF8,SjERF9,SjERF11,SjERF26,
SjERF29,SjERF30,SjERF38,SjERF44,SjERF46,SjERF49,and
SjERF51 genes exhibited a conserved homologous relationship
across all four species, suggestingthattheymightplaya
significant role in plant function.
3.6 SjERFs cluster specifically bind to the
GCC-boxes in the promoters of CEP-
associated genes in vitro
To predict the SjERFs involved in the CEP biosynthesis
pathway, NCS,6-OMT, and CNMT genes were identified in the S.
japonica genome using the BLASTP approach with p-value < 1e
-10
(Supplementary Table S2). Subsequently, the well-supported
subfamily containing the functional protein sequence was defined
as candidate functional genes in the CEP biosynthesis pathway
using a phylogenetic tree. Finally, five NCS, three 6-OMT, and five
CNMT genes were identified as candidate functional genes in S.
japonica genome (Figure 5). Meanwhile, the majority of CEP-
biosynthetic genes have high transcriptional levels in one or more
tissues of S. japonica, except for SjNCS1,SjCNMT3 and SjCNMT5
(Figure 5;Supplementary Table S8). For instance, Sj6OMT1,
SjNCS3-5, and SjCNMT4 have the highest expression level in S.
japonica root (FPKM >30), while Sj6OMT3,SjNCS2,and
SjCNMT1,2 exhibited preferential expression patterns in S.
japonica shoots.
An increasing amount of data suggests that the ERF gene
clusters play a crucial role in secondary metabolism (Paul et al.,
2020;Shoji and Yuan, 2021). The cis-acting elements of CEP
biosynthetic gene promoters were analyzed. Seven of the thirteen
FIGURE 2
Schematic diagram of phylogenetic analysis, exon/intron distribution, and motifs analysis of SjERF TFs. (A) Phylogenetic tree of 59 SjERF proteins. (B) Motif
distribution of 59 SjERF proteins. (C) The exon-intron structure of 59 SjERF genes. Yellow rectangle: UTR; black line: intron; blue rectangle: CDS.
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promoters (SjNCS1,SjNCS3,SjNCS4,SjNCS5,Sj6OMT1,Sj6OMT2,
and SjCNMT4) contained either a predicted GCC motif or a GCC-
like element (Figure 5D). Among them, three GCC-boxes were
found in the promoter region of SjNCS4 and SjCNMT4, whereas
two GCC-boxes were identified in the SjNCS5 and Sj6OMT2
promoter. To further identify the SjERFs gene cluster involved in
CEP biosynthesis, Y1H assays were carried out in this study. As
depicted in Figure 5E, binding of the AD-SjERF9/10/11 (GAL4 AD-
prey protein) fusion protein, but not AD-EV (GAL4 AD empty
vector) alone, to three tandem repeats of the GCC-box, strongly
activated the expression of the LacZ reporter gene in the Y1H
system. Moreover, the SjERF9 transcription factor regulates the
expression of SjNCS5 by directly binding the GCC-box2 of the
SjNCS5 promoter. SjERF10 could directly bind to the SjNCS5
promoter, while SjERF11 recognizes GCC-box2 of the Sj6OMT2
promoter. Interestingly, the SjERFs gene cluster was observed to
FIGURE 3
Pivotal cis−elements in the promoter of SjERF TFs.
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bind to the GCC-box1, 2 in the promoter of SjCNMT4, indicating
their potential crucial role in CEP metabolism. Additionally, the
SjERFs gene cluster-YFP fusion proteins were observed exclusively
in the nuclei, which is consistent with their putative role as
transcription factors in the nucleus (Figure 6). In conclusion, our
findings suggest that the SjERFs gene cluster regulates CEP
biosynthesis by directly binding to the GCC-boxes in the
promoters of CEP-associated genes.
3.7 Tissue expression profiling of SjERF TFs
We analyzed the expression level of 59 SjERFs in roots, stems,
leaves, and shoots of S. japonica from the available transcriptome
data. Heat-map analysis showed that eleven SjERF genes were
highly expressed in roots, stems, leaves, and shoots of S. japonica
(FPKM > 50), including SjERF5,9,17,20,29,43,45,49,51,55, and
57 (Figure 7A). Among them, SjERF20,29,43,45, and 57 showed
the highest expression level in S. japonica roots, and stems,
respectively. However, thirteen genes had nearly no expression in
the roots, stems, leaves, and shoots of S. japonica (FPKM < 1).
Furthermore, some SjERF genes with tissue-specific or preferential
expression patterns were observed in vegetative tissues of S.
japonica. For example, SjERF5 and SjERF49 with the highest
expressions were observed in S. japonica leaves. 13 SjERFs were
observed with higher expression in root tissues of S. japonica.To
validate the accuracy of RNA-seq, real-time qPCR was performed
on six SjERFs, which exhibited significantly higher expression levels
in the root of S. japonica. Overall, the results indicated that these
SjERFs exhibited higher expression levels in the roots and lower
expression in the leaves of S. japonica (Figure 7B). The qRT-PCR
results of six SjERFs were consistent with the RNA-Seq data,
indicating strong reliability of the RNA-Seq data.
3.8 Co-expression analyses of SjERFs
involved in CEP biosynthesis
Co-expression analysis of SjERFs, CEP biosynthetic genes, and
BIAs metabolites was visualized using the Cytoscape tool. The co-
expression network analysis revealed a strong correlation between
the expression levels of 35 SjERFs and CEP biosynthetic genes in S.
japonica (Pearson correlation coefficient r > 0.9 and p-value < 0.05).
It is worth noting that SjERF17 and SjERF58 were strongly
positively correlated with the three CEP biosynthetic genes,
respectively (Figure 8;Supplementary Table S9). The expression
FIGURE 4
The chromosome distribution and synteny analysis of SjERFs. (A) Chromosomal locations and their synteny of SjERFs. The connecting lines indicate
duplicated gene pairs in 59 SjERFs. (B) The phylogenetic tree of SjERFs and functional ERF cluster. (C) The topologically associating domains (TADs)
region of three SjERFs.
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profile of SjERF10 was correlated strongly with SjNCS2 and
SjCNMT2 genes. Additionally, SjERF54 was highly positively
correlated with ten BIAs, while SjERF35 was highly negatively
correlated with these metabolites, including (S)-Norcoclaurine, N-
Methylcoclaurine, 3-Hydroxy-N-methylcoclaurine, Magnoflorine,
Coptisine, (S)-Tetrahydrocolumbamine, Guattegaumerine,
Daurisoline, Fangchinoline, and Cepharanthine. SjERF42 and
SjERF52 were highly positively correlated with seven BIAs. In
summary, these findings suggest that these SjERFs may be
involved in the biosynthesis of CEP and its precursors.
4 Discussion
The AP2/ERF gene family is a plant-specificgroupof
transcription factors, characterized by an AP2 domain for DNA
FIGURE 5
Members of the SjERFs cluster specifically bind to the GCC boxes in the promoters of CEP-associated genes in vitro.(A–C) Phylogenetic tree of CEP
biosynthetic genes using MEGA11 with 1000 bootstrap replicates by Neighbor-joining (NJ) method. (D) Schematic diagrams of the SjNCS4,SjNCS5,
Sj6OMT2, and SjCNMT4 promoters. The positions of potential GCC boxes are shown as blue Rectangles. (E) Yeast one-hybrid (Y1H) assay indicates
that the SjERFs cluster binds to the GCC box in the promoters of CEP-associated genes, including SjNCS4,SjNCS5,Sj6OMT2 and SjCNMT4. Yeast
cells transformed with different combinations of constructs were grown on SD/−Ura/−Trp/+X-gal medium. Photographs were taken after 3 d of
incubation at 30°C. Y1H assays were repeated three times.
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binding (Licausi et al., 2013). Typically, members of this family
usually contain one to two highly conserved AP2 domains. The AP2
subfamily members consist of members with two repeated AP2
domains, while the ERF subfamily contains members with a single
AP2 domain (Sakuma et al., 2002). ERF transcription factors have
significant effects in regulating the biosynthesis of the main
pharmaceutical active components in medicinal plants (Gu et al.,
2017;Wang M. et al., 2021). Extensive research on the ERF family
has been conducted in various plants, including soybean, tomato,
apple, corn, barley, and common wheat (Gu et al., 2017;Feng et al.,
2020). However, genome-wide identification of ERF protein in BIA-
producing plants remains limited.
In this study, 59 SjERFs have been identified in the S. japonica
genome (Table 1;Figure 1), which is similar to the 60 EcERF genes
in E. californica (Yamada et al., 2020), 59 in Cannabis sativa (Tian
et al., 2020), and 65 in Spirodela polyrhiza (Tian et al., 2020). Each
of these ERF genes is characterized by a single conserved AP2
domain. Notably, the number of SjERF gene members in the S.
japonica genome was less than Oryza sativa (139 genes), Zea mays
(136), A. thaliana (122), Glycine max (122), and Triticum aestivum
(99) (Nakano et al., 2006;Feng et al., 2020). Collinear analysis was
performed to explore the potential relationships between the SjERF
genes in the S. japonica genome. The analysis revealed that a total of
twelve SjERF genes were involved in six segmental duplication
events (Figure 4). These segmental duplication events have likely
contributed to the expansion of the ERF family in S. japonica.
Two commonly used classification systems were established in
A. thaliana (Riechmann and Meyerowitz, 1998;Nakano et al.,
2006). Riechmann et al. classified 144 AP2/ERF transcription
factors into three classes (Riechmann and Meyerowitz, 1998);
Sakuma et al. divided 145 AP2/ERF transcription factors into five
classes and further divided the DREB subfamily into six subgroups
(A1 to A6), and the ERF subfamily into six subgroups (B1 to B6)
(Sakuma et al., 2002). In contrast, Nakano et al. classified ERF
subfamily transcription factors into ten groups, which were named
groups I to X, instead of the two major subfamilies (DREB and ERF)
(Nakano et al., 2006). A phylogenetic tree of this work showed that
59 SjERFs were further categorized into ten subfamilies based on
121 AtERFs (Figure 1). This classification was in harmony with the
evolutionary analyses of Nakano et al (Nakano et al., 2006).
Generally, ERFs within the same group exhibit evolutionary
conservation and share similar gene structures (Cao et al., 2020).
Gene structure analysis revealed that 67.8% of SjERFs, including
those from group II and III, contained only one exon, indicating a
conserved gene structure for most SjERFs (Figure 2C). These
findings were consistent with pineapple, where 66.22% of AcERFs
displayed a similar gene structure (Huang et al., 2020).
Furthermore, cis-element analysis of the promoter regions
FIGURE 6
SjERFs protein fused to a yellow fluorescent protein (YFP) transiently expressed in N. benthamiana. Scale bar: 20 mm.
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demonstrated that the majority of SjERF genes were involved in
light-responsive processes (119), phytohormone responses (ABA,
MeJA) (253), as well as abiotic and biotic stress responses (565)
(Figure 3). Specifically, 164 abscisic acid responsiveness cis-acting
regulatory elements (ABREs) were detected in the promoter regions
of 57 SjERFs, excluding SjERF57 and SjERF59. Additionally, MeJA-
responsive elements were discovered in the promoter regions of 54
SjERF genes, including SjERF gene clusters. Previous studies
demonstrated that JAs are key signaling molecules involved in
alkaloid biosynthesis (Wang M. et al., 2021). Many ERF
transcription factors respond to jasmonic acid and activate the
expression of alkaloid-associated genes, such as CrORCA and
NbERF189 (van der Fits and Memelink, 2000;Shoji et al., 2010).
Thus, these findings indicated that SjERFs can be regulated by
various cis-acting elements in their promoters during growth and
stress responses.
ERF TFs not only affect plant growth and development but also
play a crucial role in secondary metabolisms, such as terpenoids,
phenylpropanoids, and alkaloids (Zhou and Memelink, 2016;
Shoji and Yuan, 2021;Godbole et al., 2022). The majority of
ERFs shown to participate in secondary metabolites biosynthesis
are members of group IX. Several group IX AP2/ERFs form
physically linked gene clusters and have been characterized in a
limited number of plant species, including Nicotiana tabacum
(Kajikawa et al., 2017), potato (Cardenas et al., 2016), and C.
roseus (Paul et al., 2017). For instance, AaORA positively
regulates artemisinin biosynthesis in Artemisia annua and
activates the expression of AaADS,AaCYP71AV1, and AaDBR2
(Lu et al., 2013). In C. roseus, the ORCA cluster, consisting of
ORCA3, ORCA4, and ORCA5, is a crucial regulator in alkaloid
biosynthesis (van der Fits and Memelink, 2000;Singh et al., 2020). It
is also proved that ERF189, ERF221, and the NIC2-locus clustered
ERFs in N. benthamiana activate the nicotine biosynthetic pathway
by affecting several nicotine biosynthetic genes (Shoji et al., 2010).
To date, only the genome-wide identification and systematic
analysis of ERF transcription factors in E. californica,which
produces BIA, have been completed. It has been found that four
Group IX ERFs can activate the expression of key enzyme genes
involved in BIA biosynthesis (Yamada et al., 2020). In Coptis
chinensis,cis-acting elements of BIA biosynthetic gene promoters
were conducted and showed the involvement of GCC-box and ERF
transcription factors in the regulation of berberine biosynthesis
(Yamada et al., 2016). Nonetheless, the role of the ERF subfamily in
CEP biosynthesis remains unexplored. In the present study, the co-
expression network between SjERFs, CEP-associated genes, and
BIAs metabolites showed that SjERF17 and SjERF58 have a strong
FIGURE 7
Expression patterns of 59 SjERFs in different tissues of S. japonica. (A) Hierarchical clustering of the expression level of SjERFs with RNA-Seq.
(B) The expression profiles of six SjERFs in different tissues with the qRT-PCR method.
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correlation with the expression levels of CEP biosynthetic enzyme
genes. SjERF42 and SjERF52 show positive correlations with the
content of seven BIA metabolites; These results suggested that they
might be involved in regulating the biosynthesis of CEP and its
precursors (Figure 8). Notably, an ERF cluster (SjERF9/10/11) has
also been identified in the S. japonica genome and is localized to the
nucleus, respectively. Yeast one-hybrid assays proved that three
SjERFs could directly bind to several CEP biosynthetic genes,
including SjCNMT4 (Figure 5). In summary, the findings of this
study indicate that SjERF cluster may act as a direct regulator of
CEP metabolism by regulating the expression of CEP-associated
genes. This study provides a foundation for analyzing the
underlying molecular mechanism of CEP biosynthesis and further
investigating the functional genomics of candidate SjERF genes.
FIGURE 8
Analysis of correlation between SjERFs, CEP-biosynthetic genes, and BIAs metabolites. (A) Co-expression network of SjERF genes, CEP biosynthetic
genes, and BIAs metabolites with |r| > 0.9 and p-value < 0.05. Orange squares: CEP biosynthetic genes, red circles: SjERF genes. Blue octagons: BIAs
metabolites. (B) The cluster heatmap shows the expression correlations between five CEP biosynthetic genes, and two BIA precursors, alongside 23
BIA-type structures, and forty-six SjERFs.
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5 Conclusions
This is the first study that 59 SjERFs were identified and
categorized into ten subfamilies in S. japonica genome. Through a
series of bioinformatics analyses of 59 SjERFs, it was found that the
gene structure of SjERF32, and SjERF54 in same group was highly
similar. Through collinear analysis, we identified twelve SjERF
genes from the ERF genome data of S. japonica that were
involved in six segmental duplication events. One gene cluster
containing three SjERF genes was found on chromosome 2,
which is close to the evolution of functional ORCA genes in C.
roseus. Furthermore, the SjERFs cluster was observed to bind to the
CEP-associated gene promoters, suggesting that the SjERFs cluster
may act as a direct regulator of CEP metabolism. The tissue
expression profile revealed that most SjERF genes were highly
expressed in S. japonica root. Furthermore, we constructed a co-
expression network between SjERFs, CEP biosynthetic genes, and
BIAs metabolites, and several SjERFs were highly positively
correlated with the contents of diverse BIAs of S. japonica. These
results provide a basis for further characterizing the biological
function of the SjERF gene and analyzing its molecular
mechanism of regulating CEP biosynthesis.
Data availability statement
The raw data of Genome and RNA-seq data sets for the
transcriptome analysis are available in NCBI, under BioProject
PRJNA888087.
Author contributions
HY: Writing –original draft, Data curation, Investigation,
Validation, Visualization. BL: Formal analysis, Visualization,
Writing –original draft. HD: Visualization, Writing –original
draft. ZL: Visualization, Writing –original draft. XL: Resources,
Writing –review & editing. TH: Writing –original draft. YW:
Writing –original draft. YZ: Writing –original draft. CW: Funding
acquisition, Methodology, Writing –review & editing. LL:
Methodology, Resources, Writing –review & editing. SC:
Funding acquisition, Writing –review & editing. CS: Funding
acquisition, Writing –review & editing.
Funding
The author(s) declare financial support was received for the
research, authorship, and/or publication of this article. This work
was supported by introduces the talented person scientific research
start funds subsidization project of Chengdu University of
Traditional Chinese Medicine (030040015, 030040017), Sichuan
Province Innovative Talent Funding Project for Postdoctoral
Fellows (BX202206), China Postdoctoral Science Foundation
(2023M730383), and Hubei science and technology planning
project (2020BCB038).
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2024.1433015/
full#supplementary-material
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