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Min et al. BMC Plant Biology (2025) 25:560
https://doi.org/10.1186/s12870-025-06602-x BMC Plant Biology
*Correspondence:
Jinyuan Chen
20211027@qhnu.edu.cn
Shengcheng Han
schan@bnu.edu.cn
1Key Laboratory of Biodiversity Formation Mechanism and
Comprehensive Utilization of the Qinghai-Tibet Plateau in Qinghai
Province, College of Life Sciences, Qinghai Normal University, Xining,
Qinghai 810008, China
2College of Life Sciences, Beijing Normal University, Beijing 100875, China
3Academy of Plateau Science and Sustainability of the People’s
Government of Qinghai Province & Beijing Normal University, Qinghai
Normal University, Xining, Qinghai 810008, China
Abstract
Background Transcription factors (TFs) are crucial regulators of plant growth, development, and resistance to
environmental stresses. However, comprehensive understanding of the roles of TFs in speciation of Orinus, an
extreme-habitat plant on the Qinghai-Xizang (Tibet) Plateau, is limited.
Results Here, we identied 52 TF families, including 2125 members in Orinus, by methodically analysing domain
ndings, gene structures, chromosome locations, conserved motifs, and phylogenetic relationships. Phylogenetic
trees were produced for each Orinus TF family using protein sequences together with wheat (Triticum aestivum L.)
TFs to indicate the subgroups. The dierences between Orinus and wheat species in terms of TF family size implies
that both Orinus- and wheat-specic subfamily contractions (and expansions) contributed to the high adaptability
of Orinus. Based on deep mining of RNA-Seq data between two species of Orinus, O. thoroldii and O. kokonoricus, we
obtained dierentially expressed TFs (DETFs) in 20 families, most of which were expressed higher in O. thoroldii than
in O. kokonoricus. In addition, Cis-element analysis shows that MYC and G-box elements are enriched in the promoter
region of DETFs, suggesting that jasmonic acid (JA) and abscisic acid (ABA) act synergistically in Orinus to enhance
the signalling of related abiotic stress responses, ultimately leading to an improvement in the stress tolerance and
speciation adaptation of Orinus.
Conclusions Our data serve as a genetic resource for Orinus, not only lling the gap in studies of TF families within
this genus but also providing preliminary insights into the molecular mechanisms underlying speciation in Orinus.
Keywords Orinus, Phylogenetic relationship, Speciation adaptation, Transcription factor, Transcriptional regulation
Transcription factors in Orinus: novel insights
into transcription regulation for speciation
adaptation on the Qinghai-Xizang (Tibet)
Plateau
QinyueMin1, KaifengZheng2, YanrongPang2, YueFang1, YanfenZhang1, FengQiao1, XuSu1, JinyuanChen1*
and ShengchengHan2,3*
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Page 2 of 17
Min et al. BMC Plant Biology (2025) 25:560
Background
e Qinghai-Xizang (Tibet) Plateau (QTP) has a unique
geographic situation and a relatively extreme climate,
with very low temperatures, strong ultraviolet radia-
tion, and drought, and yet still has high biodiversity
[1–3]. Orinus, a xerophytic endemic alpine genus in the
family Poaceae, comprises two ecologically distinct spe-
cies: O. thoroldii (Stapf ex Hemsl.), distributed at 3300–
4500m elevation, and O. kokonoricus (K. S. Hao), found
at lower altitudes of 2500–3400m [4]. is valuable for-
age resource exhibits high stress resistance that is used to
generate high-quality agricultural varieties and improve
forage utilisation by livestock [5, 6]. On the QTP, Orinus
contributes to soil stabilisation and sand xation, which
are pivotal in ecological and conservation contexts, due
to its prolic root system and high drought, cold, and
alkali resistance [4, 7, 8]. ere have been numerous
recent reports on the adaptation of high-altitude species
to extreme habitats, including cold and drought condi-
tions [9–11]. Yet little research has examined the mecha-
nisms by which Orinus adapts to extreme environments
at high elevations. From 2024 onwards, Qu accomplished
a chromosome-scale genome assembly and annotation
for Orinus kokonoricus, a tetraploid plant (2n = 4x = 40)
and the annotation results included: 48,321 protein-
coding genes and multiple non-coding RNA types [12].
is milestone work has established O. kokonoricus as an
emerging model for studying post-polyploidization adap-
tive evolution in desert plants. Since then, research on
the adaptation of Orinus species to extreme habitats has
been initiated [13].
Speciation adaptation refers to the process through
which species develop traits that enhance their ability to
survive and reproduce in specic environments [14–16].
It is generally accepted that natural selection, genetic
variation, and environmental pressures lead to species
adaptability [17]. Research on nine genetic groups in
Rhododendron has identied genes involved in oxidative
phosphorylation, nucleotide excision repair, and biosyn-
thesis mechanisms involved in the underlying conver-
gent genomic evolution and environmental adaptation
[18]. In alfalfa, dierential expression of cold stress resis-
tance genes leads to adaptive evolution in response to the
region’s harsh cold climate [19]. Recent studies have iden-
tied that the regulatory roles of transcription factors
(TFs) in abiotic stress responses and gene family expan-
sion/contraction are two important adaptive strategies
[20]. WRKY family TFs interact with upregulated genes
involved in dierent biological processes to enhance salt
tolerance, contributing to adaptive evolution [21]. e
S-acylation cycle of MtNAC80 regulates the cold toler-
ance of Medicago truncatula [22]. ese outcomes imply
that TFs have major impacts on plant adaption to the
environment.
TFs are proteins that play essential roles in regulat-
ing plant growth, development, and resistance to abiotic
stresses [23–25]. A typical TF contains a DNA-binding
domain, transcription regulation domain, oligomerisa-
tion site, and nuclear localisation signal [26, 27]. e
functions and characteristics of TFs are determined
by these domains via interactions with cis-acting ele-
ments, and regulate target gene expression [28]. Since
2000, Riechmann analyzed the TFs of Arabidopsis thali-
ana according to the genome-wide identication [29].
It is estimated more that 5% of the Arabidopsis genome
encodes for over 1500 TFs. e large number and variety
of TFs in Arabidopsis reect the complexity of transcrip-
tional regulation in higher plants and the importance of
studying TFs [30, 31]. Some stress-related TFs modu-
late the expression of numerous stress-associated genes,
implying that TFs can be used to improve the stress resis-
tance of plants [32, 33]. Based on their DNA binding
domains, higher plants possess 64 TF families, including
the bHLH, NAC, C2H2, ERF, and MADS families [29].
ese gene families are valuable since they regulate pro-
cesses in plants [34–36]. Increasing numbers of TFs in
dierent plant species have been published in databases
such as the PlantTFDB [37], JASPAR [38], and PlantPAN
[39]. e PlantTFDB is a comprehensive TF database
for green plant genomes, and includes 320,370 TFs from
165 species and 58 families [40]. Despite these superb
resources, a thorough analysis of Orinus is lacking. Filling
the gaps regarding knowledge of the regulatory mecha-
nisms in Orinus will also facilitate investigations of TF
families in the plant kingdom.
In this study, to elucidate Orinus TFs for the broader
plant science community, we conducted a bioinformatics
analysis of 48,321 protein-coding genes from the Orinus
kokonoricus genome, with an in-depth analysis of approx-
imately 2,000 TFs. We then used RNA-Seq to compare
dierentially expressed TFs (DETFs) in Orinus thoroldii
and O. kokonoricus and predict their functions in adapt-
ing to the environment across the molecular mechanisms
of TFs. Our data will serve as a genetic resource for Ori-
nus, lling the gap in studies of TF families within this
genus and enabling preliminary insights into the roles of
transcriptional regulation in the molecular mechanisms
underlying the adaptation of Orinus on the QTP.
Results
Identication of TF families in Orinus
To characterise Orinus TF genes, an NCBI CDD search
was conducted against the Orinus genome. In all, 2125
potential TF sequences were discovered (Fig. 1A). After
examining each of the 58 TF families in Arabidopsis, we
found that 52 TF families are represented in Orinus (we
merged AP2, ERF, and RAV into the AP2/ERF family;
combined MYB and MYB-related into the MYB family;
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Min et al. BMC Plant Biology (2025) 25:560
and integrated M-type-MADS and MICK-MADS into
the MADS-box family). e accurate count of genes dis-
covered per family is depicted in (Fig.1B; Additional le
2: Table S1). e biggest TF family was bHLH, with 264
members; the AP2/ERF, bZIP, C2H2, G2-like, HB-ZIP,
MYB, MADS-box, NAC and WRKY gene families also
had many members in Orinus. e smallest families were
the HRT-like, LFY, S1Fa-like, and STAT families, which
had only two members each. Moreover, we characterised
the protein features of the TF family members. e pro-
tein lengths were 61–3023 amino acids, the molecular
weights were 7.20–323.34 kDa, and the aliphatic index
values were 31.40–108.97. In addition, 43% (913/2125) of
the proteins, including 42 TF family members, were acidic
proteins, with a pI < 7, while the remainder were basic
proteins. Notably, all the BBR-BPC, HRT-like, LSD, LYF,
NF-X1, NF-YA, S1FA-like, SRS, STAT, and Whirly TF fam-
ily members were basic proteins. Of the 2125 TFs, 100 had
an instability index < 40 and 61 had GRAVY < − 0.1, which
implies that these TFs are unstable proteins with negative
hydrophobicity (Fig.1C and D; Additional le 2: Table S2).
After identifying the TF family members, we evaluated
the size of these families compared to the Arabidopsis thali-
ana, Setaria italica, Oryza nivara, Triticum aestivum, and
Hordeum vulgare genomes. e majority of the TF families
in Orinus were comparable in size to those found in these
ve plant species, while that of eight families diered signif-
icantly on comparing our Orinus dataset with the TFs from
four complete plant genomes (Additional le 2: Table S3).
Orinus appears to have proportionally fewer B3, bZIP, C3H,
DBB, HB-other, NF-YA, Nin-like, and C2H2 genes. e
HD-ZIP gene TF family in Orinus may have more mem-
bers. Overall, the ndings indicate that the evolution of Ori-
nus is not related to the number of TF family members.
Similarities between Orinus and wheat TFs
To delve deeper into the TF gene families, phyloge-
netic trees were generated using IQ-tree with the
Fig. 1 Characterisation of TFs in Orinus. A Schematic computational pipeline for the identication of TFs in Orinus. B Distribution of Orinus TFs in 52 fami-
lies showing the minimum number of members identied in each of the 52 TF families found in the Orinus dataset. C Protein lengths of all identied TFs
in Orinus. D Isoelectric point (pI) of all the identied TFs in Orinus
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Min et al. BMC Plant Biology (2025) 25:560
maximum-likelihood method. In every instance, the
conserved domains served as the basis for construct-
ing the phylogenetic trees. Two phylogenetic trees were
constructed for the TF families, one comprising only
the Orinus TF family members and the other combining
the Orinus sequences and chosen sequences from wheat
(Triticum aestivum L.) TF families that function as mark-
ers for unique subgroups. We used the wheat domains
because wheat is a major food crop that serves as a model
species to an extent. e wheat genome is huge and com-
plex, containing three subgenomes, namely A, B, and
D. is complexity oers abundant information and is
ideal for studying genome structure, gene function, and
the evolution of polyploid plants, such as Orinus species
(2n = 4x = 40) [41]. en, we analysed 10 TF families in
Orinus: AP2/ERF, bHLH, C2H2, GRAS, HD-ZIP, MADS-
box, R2R3-MYB, NAC, TALE, and WRKY (Additional
le 2: Tables. S4-S13).
The AP2/ERF family
Our study identied 148 members of APETALA2/eth-
ylene response factor (AP2/ERF) family containing the
highly conserved DNA-binding domain (Additional
le 2: Tables. S4, Additional le 1: Fig. S2A). In addi-
tion to the 10 known subfamilies divided in Arabidopsis,
we subdivided IV into IVa and IVb and identied a new
subfamily, named XI, which consists of 32 members, mak-
ing it the largest subfamily within the AP2/ERF family
(Fig.2). Meanwhile, the XI members possess highly spe-
cic conserved motifs that distinguish them from other
Fig. 2 Phylogenetic tree of AP2/ERF genes in Orinus and wheat. The phylogenetic tree was generated using the maximum-likelihood approach, based
on the alignment of the AP2/ERF domains. Only bootstrap values exceeding 50% are shown (calculated with 1000 replicates to verify the reliability of the
tree topology). These AP2/ERF proteins are clustered into 13 clades. The red star indicates the new subfamily discovered in Orinus
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Min et al. BMC Plant Biology (2025) 25:560
subfamilies (Additional le 1: Fig. S2A, S4A). However, no
VI subfamily members were detected in wheat, suggesting
subfamily contraction during wheat evolution. And the
new subfamily XI is probably involved in adaptation to the
distinct growth environment of Orinus, such as by acquir-
ing new functions to enhance drought and cold resistance.
The bHLH family
Genome-wide analysis revealed 264 putative basic helix-
loop-helix (bHLH) protein family. In wheat, bHLH is
divided into 23 subfamilies [42]. Compared with the 23
clearly dened subgroups found in wheat, we discovered
two new subgroups in Orinus named IIIg and XVI, while
IIIf was absent (Fig. 3). is implies that these proteins
have special functions that were either lost in wheat and
Arabidopsis or acquired after the divergence of Orinus
from the last common ancestor. Conserved motif analy-
sis revealed distinct patterns (motif 1,2 and 6) among XVI
subfamily members, suggesting subfamilies have func-
tional divergence (Additional le 1: Fig. S2B, Fig. S4B).
ese results also revealed that apart from the bHLH
domain, there is no overall collective amino acid sequence
in bHLH proteins (Additional le 1: Fig. S2B, S4B).
The C2H2 family
C2H2 zinc nger proteins are a diverse group of TFs that
play critical roles in the regulation of gene expression
in eukaryotic cells [43]. Phylogenetic analysis identied
Fig. 3 Phylogenetic tree of bHLH genes in Orinus and wheat. The phylogenetic tree was generated using the maximum-likelihood approach, based on
the alignment of the bHLH domains. The parameters are consistent with Fig.2. These bHLH proteins are clustered into 25 clades. The red star indicates the
new subfamily discovered in Orinus and the blue star indicates the subfamily that is missing in Orinus
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Min et al. BMC Plant Biology (2025) 25:560
total 77 members in Orinus and two new subfamilies (IIb
and IIIb) in Orinus and wheat (Fig.4, Additional le 1:
Fig. S2C, S4C). Diversication of C2H2 genes can lead
to loss of their original functions or gain of new func-
tions to boost the adaptability of plants [44, 45]. Recently,
research on plant C2H2-type zinc nger proteins has
increased both domestically and internationally, and
C2H2-type zinc nger proteins related to plant growth,
development, and stress tolerance have been discov-
ered [46, 47]. is implies that members of IIb and IIIb
undergo dierentiation between monocotyledons and
dicotyledons and may possess distinct functions in spe-
ciation adaptation.
The GRAS family
GRAS proteins have been categorised into 8–17 subfami-
lies in species such as Arabidopsis [48], Populus tricho-
carpa [49], Citrus sinensis [50] and some angiosperm
species that show maximum divergence [51]. In this
study, the 61 Orinus GRAS genes were classied into 10
clades, SHR, PAT1, OS19, SCR, SCL4/7, HAM, DELLA,
SCL4, LISC1, and DTL, two less than the number of
wheat subfamilies [52] (Fig. 5). Phylogenetic analysis
Fig. 4 Phylogenetic tree of C2H2 genes in Orinus and wheat. The phylogenetic tree was generated using the maximum-likelihood approach, based on
the alignment of the C2H2 domains. The parameters are consistent with Fig.2. These C2H2 proteins are clustered into ve clades. The red stars indicate
the new subfamilies discovered in Orinus
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Min et al. BMC Plant Biology (2025) 25:560
of sequence alignments coupled with motif scanning
showed GRAS family members have conserved C-termi-
nus and variable N-terminus (Additional le 1: Fig. S2D).
Since previous studies proposed that GRAS proteins
might have undergone expansion following the diver-
gence of higher and lower plants, with their numbers
showing signicant variation across plant species, and
some species or lineages potentially maintaining partic-
ular GRAS subfamilies as evolution progressed [53, 54].
en the results indicated that the Orinus GRAS protein
subfamily may experience some degree of dierentiation
between monocotyledons and dicotyledons. Gene struc-
ture analysis found that Orinus GRAS genes possess the
motif and domain structural characteristics of this family
in other species (Additional le 1: Fig.2D, S4D). Addi-
tionally, the reduced conservation of GRAS domains
across dierent subfamilies, contrasting the increased
sequence similarities within subfamilies, sheds light on
the similar functions of GRAS genes.
The HD-ZIP family
e HD-ZIP proteins are classied into four clusters (I–
IV) based on the conservation of the HD-ZIP domain,
gene structures, other conserved motifs, and their dis-
tinct functions [55] while the 70 Orinus HD-ZIP mem-
bers belong to these four well-dened subfamilies
Fig. 5 Phylogenetic tree of GRAS genes in Orinus and wheat. The phylogenetic tree was generated using the maximum-likelihood approach, based on
the alignment of the GRAS domains. The parameters are consistent with Fig.2. These GRAS proteins are clustered into 12 clades. The blue stars indicate
the subfamilies that are missing in Orinus
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Min et al. BMC Plant Biology (2025) 25:560
(Additional le 2: Fig. S1A), which have conserved basal
architectures (Additional le 1: Fig. S2E, S4E). Sub-
families I and II exhibit closer phylogenetic relatives
than other HD-ZIP subfamilies because they both com-
posite motifs consisting of 1, 2 and 3. Meanwhile, the
phylogenetic reconstruction revealed Subfamily IV as
the most motif-rich clade. e observed patterns may
indicate strong purifying selection on essential func-
tional domains, supported by the ancestral origin of the
subfamily.
The MADS-box family
e plant MADS-box TF family can be subdivided into
type I, with a MADS domain (M domain), and type II,
with a conserved four-domain structure called “MIKC”
[56]. e 77 MADS-box proteins in Orinus can be clus-
tered into 12 subfamilies (Fig.6). ree of the clusters are
type-I MADS-box proteins. Compared to species such as
wheat and Arabidopsis, Orinus has relatively few MADS-
box proteins, and the number of type-II subfamilies is
also signicantly reduced [31, 57]. is may be related to
the high duplication rate of the MADS-box family TFs
in Orinus or the high rate of loss after duplication. ese
results indicate that there are signicant dierences in
the MADS-box family of TFs among species, with each
having distinct functions and dierent evolutionary con-
straints. While shared motifs indicate common ancestry,
the MADS-Box subfamily-specic motif combinations
Fig. 6 Phylogenetic tree of MADS-box genes in Orinus and wheat. The phylogenetic tree was generated using the maximum-likelihood approach, based
on the alignment of the MADS-box domains. The parameters are consistent with Fig.2. These MADS-box proteins are clustered into 13 clades. The blue
star indicates the subfamily that is missing in Orinus
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Min et al. BMC Plant Biology (2025) 25:560
correlate with predicted functional specializations (Addi-
tional le 1: Fig. S2F, S4F).
The R2R3-MYB family
Previous empirical studies exhibit the family is classied into
nine clusters (C1-C9) based on the 25 subfamilies in Ara-
bidopsis [58, 59]. We found that the family also comprised
nine clusters in Orinus (Additional le 1: Fig. S1B), implying
a common ancestor before the divergence of monocots and
dicots. Multiple members of the wheat R2R3-MYB fam-
ily are genes that respond to drought or high temperature.
eir functions in wheat should provide more details for
function identication in Orinus. R2R3-MYB proteins clus-
tering together within the same clade generally have simi-
lar motif compositions and all Orinus R2R3-MYB proteins
contained Motif 1, 2, 3, which form MYB DNA-binding
domains (Additional le 1: Fig.S2G, S4G).
The NAC family
e NAM, ATAF and CUC (NAC) subfamilies a–h in Ori-
nus, wheat and Arabidopsis [60], were delineated based on
prior research on subfamilies in barley [61] and rice [62],
which possess these eight main groups (Additional le 1:
Fig. S1C-1, S1C-2, S2H and S4H). Contrary to expectations
from wheat (which exhibits a contracted Subgroup f), sub-
group h is the smallest in the Orinus NAC family. Further
experimental validation is needed to uncover the family-
specic reduction. Noteworthy is the fact that group b
was divided into two subclades unveiled their topology
is complex, demonstrating multiple duplication events,
before and/or after polyploidization of Orinus (Additional
le 1: Fig. S1C-2) Moreover, each subgroup has conserved
motifs and a diverse number of proteins, probably attrib-
utable to divergent genomic events throughout the evolu-
tionary history of Orinus and other species.
The TALE family
We found 34 TALE homeodomain superclass in Orinus
in unambiguously dened subgroups (Additional le 1:
Fig. S1D) same like TALE subfamilies of sweet orange
[63], soybean [64], and tomato [65], implying that the
relationships within the TALE gene families of Orinus are
similar to those of other species. ese subgroups were
divided by homeodomain superclass encompasses the
KNOTTED-like homeodomain (KNOX) and BEL1-like
homeodomain (BELL) proteins and these two groups
share structural and functional similarities (Additional
le 1: Fig.S2I, S4I).
The WRKY family
WRKY proteins are sorted into seven groups according
to their N-terminus amino acid sequences and the archi-
tecture of their distinct zinc nger motifs at the C-termi-
nus [66, 67]. We found 136 WRKY genes in the Orinus
genome, categorised into these seven distinct groups
(Additional le 1: Fig. S1E, S2J and S4J), comparable to
the groups in wheat [68]. However, because wheat is
hexaploid and possesses three subgenomes, wheat should
theoretically possess 1.5 times as many WRKY genes
as Orinus. ese results imply that the Orinus WRKY
family is evolutionarily conserved, but several specic
subgroups have expanded. e existing classication of
WRKY genes has seven categories that are well delin-
eated in wheat and various other plants. is points to
the conservation of the classication of Orinus WRKY
genes. In Arabidopsis, WRKY family members are
involved in various physiological programs in plants [69,
70]. Simultaneously, the WRKY DNA-binding domain
is highly conserved, although the overall structures are
notably divergent, which implies that subfamily members
are also functionally conservative.
Chromosomal distribution and intraspecic analysis of the
10 TF families
As genomes evolve, their broad syntenic relationships
at the gene level illustrate the tight evolutionary ties and
widespread rearrangement events involving chromo-
somes [71]. Based on the corresponding chromosome-
level genome sequence and annotation information, the
10 TF families were mapped on the 20 Orinus chromo-
somes (Additional le 1: Fig. S3A-J). Note that three
members of the bHLH family were located on unan-
chored scaolds and were not mapped (Additional le
2: Table S5). e distributions of the 10 TF families on
the chromosomes diered and were disproportionate.
Notably, only the bHLH, NAC, R2R3-MYB. MADS-box,
and WRKY families were present on every chromo-
some, although their distributions were uneven. Most
TF genes were found at the ends of chromosomes, with
fewer in central positions. Tandem and segmental dupli-
cations have been reported to play roles in the expansion
and functions of gene families [71]. is study identied
intraspecic syntenic blocks in each family (Fig. 7A).
ere were 155, 226, 73, 65, 69, 57, 99, 157, 36, and 140
syntenic gene pairs (Fig.7B) containing 117, 218, 62, 54,
67, 57, 80, 116, 28, and 118 homologous genes, respec-
tively (Fig.7C), in the 10 TF families. Analysing tandem
duplication events, none were found in HD-ZIP, while the
other families had one to six tandem repeat gene pairs
(Additional le 1: Fig. S3A–J). We found the number of
homologous genes is consistent with the relative abun-
dance of TF family members, whereas no such correla-
tion was observed for tandem duplications. ese results
indicate that the Orinus genome underwent a large chro-
mosome rearrangement during its evolution and seg-
mental duplications represent the dominant mechanism
of gene family expansion in Orinus, potentially serving as
a key evolutionary force.
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Min et al. BMC Plant Biology (2025) 25:560
Cis-acting element analysis in the promoter region of
dierentially expressed TF (DETF) genes
Focusing on the potential biological roles of TFs in Ori-
nus, the expression patterns of all TF families in the two
Orinus species (O. thoroldii and O. kokonoricus) grow-
ing at their characteristic altitudes were investigated
using public RNA-Seq data [13]. We found 55 predicted
dierentially expressed TFs (DETFs) in 19 TF families
with cis-acting elements in the 2-kb promoter region
upstream (Fig.8A; the FPKM values are listed in Addi-
tional le 2: Tables S4–S13). e predicted cis-acting
elements were categorised into three dierent groups
Fig. 7 Intraspecies synteny analysis of 10 TF families. A Intraspecies synteny analysis in AP2/ERF, bHLH, C2H2, GRAS, HD-ZIP, MADS-box, R2R3-MYB, NAC,
TALE, and WRKY (1–20 represent LG1–LG20, the 20 chromosomes in Orinus). B Homologous gene pairs in these 10 families. C The numbers of homologous
genes in these 10 families
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Min et al. BMC Plant Biology (2025) 25:560
(stress, hormonal, and growth) (Fig. 8B–D) and all the
predicted cis-acting elements were more likely to appear
frequently in TFs that were expressed higher in O. thor-
oldii. Among the DETFs, the types of cis-acting elements
involved in biotic and abiotic stresses were the same and
were most abundant in MYB, MYC, and STRE. Regard-
ing phytohormone responsive TFs, the TCA-element was
found only in O. thoroldii TFs that were highly expressed,
while CARE was present only in TFs that were highly
expressed in O. kokonoricus; however, the enrichment of
Fig. 8 Distribution of cis-acting elements in the 2.0-kb promoter regions of DETFs in Orinus. A Expression heatmap: expressed more in O. thoroldii
than in O. kokonoricus or vice versa. B The existence of abiotic and biotic stress-related elements in the promoter regions of DETFs. C The existence of
phytohormone-responsive-related elements in the promoter regions of DETFs. D The existence of plant-growth- and development-related elements in
the promoter regions of DETFs
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Min et al. BMC Plant Biology (2025) 25:560
both was not signicant. Note that the plant growth and
development category with the fewest numbers but the
most variance. G-box diered enormously between the
two expression patterns. Interestingly, MYC as a mem-
ber of the bHLH subfamily, binds with the HLH domain
and G-box to activate the jasmonic acid (JA) signalling
pathway, while G-box can participate in the response to
light by binding bZIP and bHLH [72, 73]. Moreover, the
AREB/ABF subfamily of bZIP can activate the abscisic
acid (ABA) signalling pathway by recognising the ABRE
element, while bZIP is also involved in the Nrf2-ARE
pathway to modulate cell defence against oxidative dam-
age from toxic substances [74]. Stomatal aperture regula-
tion by MYB involves the ABA signalling pathway [75].
We also found that the enrichment patterns of the cis-
acting elements involved in regulating the two hormones
were consistent. erefore, we believe that JA and ABA
make great eects in Orinus and may work synergisti-
cally to enhance the signalling of related abiotic stress
responses, thereby improving the stress resistance of
plants. e major regulating TF families were bHLH,
bZIP, and MYB. We are convinced that TF families such
as NAC, TALE, and WRKY, which have minimal or no
direct binding sites on cis-acting elements, exert their
roles in the downstream response in signal transduction.
Discussion
In natural environments, plants face abiotic stresses
such as drought, salt stress, and temperature, and biotic
stresses including pathogens and pests, which aect their
growth and development [76, 77]. TFs are important
regulators of stress-responsive genes and can be used to
improve crop stress resistance [78, 79]. e TFs that have
been extensively studied include NAC, MYB, WRKY,
bHLH, and AP2/ERF, and their importance in responses
to abiotic and biotic stresses by binding to cis-acting
elements in the promoter region to regulate the expres-
sion of target genes is widely recognised [80, 81]. e
promoter regions upstream of gene coding sequences
contain many cis-acting elements [82, 83]. cis-Acting ele-
ments have many functions and respond to pathogens,
wounds, light, and phytohormones. Research on cis-act-
ing elements is crucial to gaining a deeper understanding
of plant defence mechanisms in response to both abiotic
and biotic stresses [84].
Given the crucial role of TFs in plants under stress, we
annotated the O. kokonoricus genome to identify TFs.
Based on the family assignment rules, 2125 TFs were
identied and classied into 52 families, the same num-
ber as in wheat and many other monocotyledons. Nota-
bly, the SAP and NLL/SPL gene families have been little
explored in monocotyledons compared to dicotyledons.
We found no members of these two families in Orinus,
implying that monocotyledons experienced dierent
selective pressures and functional adaptations during
their evolution. For instance, the SPL gene family plays a
signicant role in owering and development in dicotyle-
dons, while its role in monocotyledons is not prominent
or may be lled by other genes [85–88]. e absence of
some gene families in monocotyledons implies that gene
families have dierent evolutionary patterns and biologi-
cal functions in monocotyledons and dicotyledons.
Phylogenetic trees generated using the Orinus and
wheat AP2/ERF, bHLH, C2H2, GRAS, HD-ZIP, MADS-
box, R2R3-MYB, NAC, TALE, and WRKY TF family
members were divided into 13, 24, 5, 10, 4, 12, 9, 8, 6,
and 7 subfamilies, respectively, with most family mem-
bers assigned to known subfamilies. During evolution,
however, the AP2/ERF and C2H2 TF families in Orinus
developed new subfamily branches, while the GRAS and
MADS-box TF families were found to exhibit subfam-
ily loss, and the bHLH gene family exhibits both above
phenomena. e formation of new subfamilies may
be accompanied by the dierentiation and innovation
of gene functions [89]. New subfamily members may
acquire functions that dier from those of the original
family members, to adapt to specic physiological needs
or environmental conditions. As previously described,
dierent subfamilies of the AP2/ERF, C2H2, and bHLH
gene families are involved in regulating dierent growth
and development processes or responding to dierent
extreme environmental stresses [90–92]. Considering the
unique environment of the QTP, we speculate that the
emergence of new subfamilies enabled Orinus to adapt
to the extreme environment and provide stronger pro-
tective mechanisms. Numerous studies have shown that
the GRAS TF family has high species specicity and its
classication into dierent subfamilies is still not stan-
dardised, with considerable divergence among the GRAS
family genes [93, 94]. MADS-box genes function in the
development of plant oral organs [95, 96]. Orinus grows
at altitudes of 2500–4000m and is exposed to diverse
environmental changes. We believe that the loss of GRAS
and MADS-box subfamilies in Orinus is a normal phe-
nomenon. Moreover, we explored the role of the bHLH
subfamily IIIf. Evidence from past studies implies that the
IIIf subfamily of the bHLH family in plants is involved in
regulating the synthesis of anthocyanin and some sec-
ondary metabolites [97]. e absence of subfamily IIIf
in the bHLH family of Orinus implies that plants on the
QTP have evolved more environment-responsive genes.
is family-specic expansion/reduction requires further
investigation to determine its biological relevance.
Tandem and segmental duplications, which are central
to species evolution and the expansion of gene families,
are the primary forces driving the emergence of new gene
functions [71, 98]. Consequently, we focused on the TF
families that have been identied as being important in
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 13 of 17
Min et al. BMC Plant Biology (2025) 25:560
abiotic stress. Interspecic collinearity analysis of 10
TF families detected 1077 collinear gene pairs in Ori-
nus, but only 25 pairs of tandem duplicated genes. e
collinear relationships were not as impressive as we had
hoped. Based on phylogenetic relationships, one reason-
able explanation is that the Orinus genome underwent a
large chromosome rearrangement during its evolution
[99]. Simultaneously, the motif analyses indicated that
homologous gene pairs from the same clade shared con-
served motif distributions that corresponded to a spe-
cic function, as found in other studies [100, 101]. Still,
we believe that tandem and segmental duplications are
the most frequent expansion mechanisms for TF families
that might lead to neofunctionalisation, subfunctionalisa-
tion, or specialisation as evolution progresses, inuenc-
ing functional divergence. A persistent duplication event
in Orinus might have facilitated the divergence of all the
TF families.
Cis-acting elements inuence the regulation of gene
expression under stress responses in organisms [102].
e DRE core regulates gene expression under abiotic
stress, such as drought, high salinity, and low tempera-
ture [103]. e TCA-element and CARE are involved
in the respective salicylic acid (SA) and gibberellic acid
(GA3) responses in seeds [104, 105]. Moreover, the cis-
acting elements we identied that are involved in plant
growth and development are part of a conserved DNA
module involved in light responsiveness [106]. e G-box
is present in TFs that are expressed higher in O. thoroldii.
e G-box is specically bound and regulated by the GBF
family of bZIP proteins to mediate blue-light-induced
photomorphogenesis, and ABA and JA signalling pro-
motes a reactive oxygen species (ROS) burst under envi-
ronmental stress [107, 108]. e results of our analysis
of cis-acting elements imply that JA- and ABA-related
elements play leading roles in Orinus transcriptional
regulation, with many elements chained together to func-
tion. Much research has highlighted the pivotal roles of
JA and ABA in enhancing plant resilience under various
abiotic stresses [109–111]. Our ndings also imply that
the signalling pathways of these two hormones may act
in concert to enhance the adaptability of the two Orinus
species to the environment of the QTP. e dierential
expression analysis between O. thoroldii and O. kokonori-
cus shows that both play roles in adaptation to extreme
habitats, but more genes involved in adaptive regulation
are expressed in O. thoroldii. Further research and explo-
ration needed to elucidate the synergistic functions of JA
and ABA.
TFs are major drivers of evolution and domestication,
and they can potentially be used to improve crops and
ne-tune traits [112]. Our study included an initial analy-
sis of 52 Orinus TF families and helps to ll the gap in
research on TF families across the plant kingdom. e
results should help to elucidate the adaptation of Orinus
to extreme environments and provide new understanding
of the speciation of O. thoroldii and O. kokonoricus from
the perspective of TFs. Our dataset is the most extensive
collection of TF sequences from any Orinus species.
Conclusions
is study identied 2125 TF genes in Orinus, dispersed
across 20 chromosomes. Further analysis revealed that
the conserved motifs and gene structures were simi-
lar within the same subfamilies, implying a high degree
of gene conservation. Homology analysis indicated that
tandem duplication and segmental duplication were driv-
ing forces in the evolution of TF subfamilies in Orinus.
An analysis of cis-acting elements on DETFs found that
the Orinus TF families had more hormone-response ele-
ments like ABRE and-stress defence elements like MYB,
MYC, and STRE indicated that JA and ABA may contrib-
ute to speciation adaptation. e TFs identied here pro-
vide new insights into the evolutionary adaptation of O.
thoroldii and O. kokonoricus and expand the database of
TFs in the plant kingdom.
Methods
Identication of Orinus TFs
First, 48,321 protein-coding sequences (PCGs) of Ori-
nus were downloaded from the National Genomics Data
Centre, under accession PRJCA018722 [12]. To identify
the PCGs that encode TFs in Orinus, searches were con-
ducted using the sequences of conserved domains from
members of 58 TF families previously identied in Ara-
bidopsis thaliana that are listed in the PlantTFDB. en,
we put the ltered sequences into BLASTP and used the
Swiss-Prot database to nd those with the highest e-val-
ues whose recname corresponded to a TF family member
[113]. e remaining sequences were scanned using the
National Center for Biotechnology Information (NCBI)
Conserved Domain Database (CDD) to conrm the TF
domains [114]. In parallel, MEME SUITE was used to
discover novel patterns to nd conserved motifs of TF
families [115]. Subsequently, we used the amino acid
sequences of Arabidopsis to produce a phylogenetic tree
with the sequences of Orinus TF family members, using
MEGA-X to remove those with problematic evolutionary
relationships [116]. After removing redundant and erro-
neous sequences, we analysed the remaining candidate
genes.
Chromosome location, collinearity analysis, characteristic
information
e chromosomal locations, the intraspecic synteny
analyses and annotation and visualization of identied
motifs were visualized utilizing the TBtools software
[117]. e number of amino acids, molecular weight
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 14 of 17
Min et al. BMC Plant Biology (2025) 25:560
(MW), isoelectric points (pI), instability index, aliphatic
index, and grand average of hydropathicity (GRAVY)
of each TF protein were calculated using ExPASy tools
and the predicted protein subcellular localisation was
obtained on the WoLF PSORT online site [118, 119].
Phylogenetic analysis and classication of TF families
Multi-sequence alignments were performed using Mus-
cle Wrapper software to identify the same TF subfamilies
in Orinus and wheat, and an unrooted phylogenetic tree
was built under maximum likelihood with TBtools using
IQ-tree (1000 bootstraps, plus default parameters) [120].
e preliminary phylogenetic tree was imported into
iTOL online and dierent subfamilies were highlighted in
distinct colours [121].
Dierential expression analysis
e expression patterns of all the TF families in the two
Orinus species at dierent altitudes were investigated
using public RNA-Seq data [13]. Signicant DETFs in
the two Orinus species were determined based on FPKM
values. Our screening criteria were standard|log2 (fold
change)| values ≥ 1 and p-values < 0. 05.
Cis-acting elements analysis
e sequences of the DETF promoter regions (2000bp
upstream of the translational start sites of genes) were
searched using PlantCARE to identify cis-acting elements
[122].
Supplementary Information
The online version contains supplementary material available at h t t p s : / / d o i . o r
g / 1 0 . 1 1 8 6 / s 1 2 8 7 0 - 0 2 5 - 0 6 6 0 2 - x.
Supplementary Material 1
Supplementary Material 2
Acknowledgements
This work was supported by the Science and Technology Department of
Qinghai Province of China (Program No. 2023-ZJ-706), National Natural
Science Foundation of China (Grant No. 32360305), and Qinghai “Kunlun
Talents • High End Innovation and Entrepreneurship Talents” Featured Project
to Xu Su and Shengcheng Han. The funders had no role in the study design,
data collection and analysis, decision to publish, or preparation of the
manuscript.
Author contributions
QM and KZ developed the analysis approach and conceived the concept
of the manuscript. QM also wrote the manuscript. YP, YF, YZ, FQ and XS
participated in data collection and analysis. JC and SH supervised writing and
editing. All authors read and approved the manuscript.
Funding
This work was supported by the Science and Technology Department of
Qinghai Province of China (Program No. 2023-ZJ-706), National Natural
Science Foundation of China (Grant No. 32360305), and Qinghai “Kunlun
Talents • High End Innovation and Entrepreneurship Talents” Featured Project
to Shengcheng Han.
Data availability
Data is provided within the manuscript or supplementary information les.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
All authors approved the nal manuscript and the submission to this journal.
Competing interests
The authors declare no competing interests.
Received: 14 March 2025 / Accepted: 22 April 2025
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