Article

Genome-Wide Identification and Expression Pattern Analysis of

SBP Gene Family in Neolamarckia cadamba
Linhan Tang, Keying Li, Chuqing Cai, Wenjun Wu, Guichen Jian, Ziming Lei, Changcao Peng and Jianmei Long *

Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, College of
Forestry and Landscape Architecture, South China Agricultural University, Guangzhou 510642, China;
tanglinhan0721@163.com (L.T.); crel8235@163.com (K.L.); caichuqing1224@163.com (C.C.);
xh1091848031@163.com (W.W.); gustave017@163.com (G.J.); 19881774796@163.com (Z.L.);
ccpeng@scau.edu.cn (C.P.)
* Correspondence: longjianmei@scau.edu.cn

Abstract: Background: SQUAMOSA promoter-binding protein (SBP) genes encode a group

of plant-specific transcription factors that play crucial roles in plant growth, development,
and stress responses. To date, SBP genes have been reported in a number of plant species,
but the SBP gene family has not been identified in Neolamarckia cadamba, an important
fast-growing species referred to as a ‘miracle tree’ and recognized for its potential medic-
inal value in Southeast Asia. Methods: Bioinformatics approaches were employed to
conduct a comprehensive analysis of the NcSBP gene family, including investigations into
physicochemical characteristics, phylogenetic relationships, gene structure, chromosomal
localization, conserved motifs, cis-acting elements, and expression patterns. Results: A
total of 27 NcSBP members were identified in the N. cadamba genome, encoding proteins
ranging from 148 to 1038 amino acids in length, with molecular weights between 16,714.34
and 114,331.61 Da. They were classified into eight clades according to phylogenetic anal-
ysis, and unevenly distributed across 17 chromosomes, with 4 tandem gene duplication
pairs and 27 fragment duplication events. In addition, cis-acting elements associated with
hormone and light responses were most presented in the promoters of NcSBP genes. The
transcript levels of NcSBP were investigated through RNA-seq and qRT-PCR, indicating
distinct expression patterns across various tissues and under different hormone and stress
conditions. Conclusions: In summary, this study comprehensively identified and charac-
Academic Editor: Jacqueline Batley
terized the SBP gene family in N. cadamba, providing a significant foundation for further
Received: 4 March 2025 functional investigation into NcSBP genes.
Revised: 10 April 2025
Accepted: 15 April 2025
Keywords: Neolamarckia cadamba; SBP; expression pattern; bioinformatic analysis
Published: 17 April 2025

Citation: Tang, L.; Li, K.; Cai, C.; Wu,

W.; Jian, G.; Lei, Z.; Peng, C.; Long, J.
Genome-Wide Identification and
1. Introduction
Expression Pattern Analysis of SBP
Gene Family in Neolamarckia cadamba. The SQUAMOSA promoter-binding protein (SBP) gene family belongs to a class of
Genes 2025, 16, 460. https://doi.org/ plant-specific transcription factors that are widely distributed in higher plants [1]. They
10.3390/genes16040460 are characterized by an SBP domain consisting of 76 amino acid residues and contain two
Copyright: © 2025 by the authors. typical zinc finger structures and a nuclear localization signal (NLS) at the C-terminus [2].
Licensee MDPI, Basel, Switzerland. The conserved SBP domain of SBP transcription factors has been demonstrated to be
This article is an open access article essential for binding to the palindromic GTAC core motif [3]. The SBP gene was first
distributed under the terms and
identified in Antirrhinum majus by isolating AmSBP1 and AmSBP2, which could bind to
conditions of the Creative Commons
the promoter of a floral meristem identity gene, SQUAMOSA [4]. The SBP genes were
Attribution (CC BY) license
(https://creativecommons.org/
subsequently identified in Arabidopsis thaliana and Zea mays, and named SBP-like genes
licenses/by/4.0/). (SPL) [5,6]. To date, SBP genes have been identified across a diverse range of species,

Genes 2025, 16, 460 https://doi.org/10.3390/genes16040460

Genes 2025, 16, 460 2 of 20

including algae, mosses [7], gymnosperms, and angiosperms [8]. However, these genes
have not been found in prokaryotes, fungi, or animals [9].
Members of the SBP family are recognized for their role in regulating various aspects
of plant growth and development, such as shoot and leaf development [10], flowering [11],
fertility, and epidermis formation [12,13]. In A. thaliana, AtSPL9 is involved in the formation
of the epidermis on both the main stem and on the inflorescence, and it plays a role in
regulating the vegetative-to-floral transition, as well as in anthocyanin accumulation [14].
AtSPL2, AtSPL10, and AtSPL11 play vital roles in the regulation of leaf morphology, shoot
maturation, and the promotion of trichome formation [15]. In addition, SPL is the target of
microRNA156 (miR156), and the miR156-SPL module regulates a range of physiological
and biochemical processes. For example, the miR156-AtSPL3 regulatory module has
been demonstrated to participate in mediating both vegetative phase transition and floral
induction processes [16]. AtSPL8 serves a dual function in the local regulation of certain
developmental processes mediated by gibberellins (GAs): it is positively regulated in GA-
mediated anther development, whereas it is negatively regulated in seedlings [17]. In maize,
the SBP transcription factor tsh4 is associated with bract development and the establishment
of meristem boundaries [6]. Furthermore, accumulating evidence has indicated the crucial
involvement of SBP genes in regulating fruit development and crop yield. More than half
of rice (Oryza sativa) OsSPL is specifically expressed in young panicles [18]. Notably, the
SPL gene TaSPL16 from wheat (Triticum aestivum), which is predominantly expressed in
developing panicles, has been shown to significantly enhance seed yield [19]. Additionally,
TaSPL21-6D-HapII contributed to a remarkable 9.73% increase in 1000-grain weight [20]. In
grapes, 12 VvSBPs from grape (Vitis vinifera) genes were expressed at a high level during
early fruit development [21]. Particularly, the tomato (Solanum lycopersicum) SBP gene
LeSPL-CNR (Colorless Non-Ripening) has been characterized as a key regulator of fruit
ripening; the methylation-mediated epigenetic modification in its promoter region leads to
the inhibition of the ripening process [22].
In addition, extensive studies have revealed that the SBP genes serve as critical regula-
tors in modulating hormone signaling pathways and orchestrating adaptive responses to di-
verse abiotic stresses in multiple plant species. For example, the interaction between DELLA
protein and AtSPL9 was obstructed by GA, leading to early flowering in A. thaliana [23].
Functional characterization revealed that OsSPL10 plays an important role in drought stress
response via the direct transcriptional regulation of the NAC (for NAM, ATAF1/2, and
CUC2) transcription factor OsNAC2, consequently modulating reactive oxygen species
(ROS) homeostasis [24]. The overexpression of BpSPL9 enhances the active oxygen scaveng-
ing ability of salt stress and drought stress by increasing the accumulation of superoxide
dismutase (SOD) and peroxidase (POD) in transgenic lines [25]. The overexpression of
an SBP gene (VpSBP16) from the Chinese wild grapevine Vitis pseudoreticulata improves
tolerance to salt and drought stress during seed germination, as well as in seedlings and
mature plants, by modulating the salt overly sensitive (SOS) and ROS signaling pathways
in transgenic A. thaliana [26]. Moreover, SBP genes are important regulators in copper
homeostasis. AtSBP7 could bind to the core elements of GTAC associated with copper
reactions, and the overexpression of AtSBP7 could reduce plant toxicity in response to Cu
and Cd [27,28]. The overexpression of OsSBP9 can enhance the accumulation of Cu in rice
seeds, thus improving digestibility and metabolism [29].
N. cadamba is an important timber tree in Southeast Asia; it is famously known as a
‘miracle tree’ due to its super rapid growth [30]. The swift advancement of next-generation
sequencing technologies has led to the identification of SBP gene families across numerous
plant species [31–33]. However, no systematic identification or characterization of SBPs has
been conducted in N. cadamba. The genome sequencing of N. cadamba has been completed,
Genes 2025, 16, 460 3 of 20

which can provide an opportunity to identify all SBP genes in N. cadamba [34]. In this study,
we employed comprehensive bioinformatics approaches to systematically identify and
characterize the SBP gene family in N. cadamba, including phylogenetic classification, gene
structure analysis, chromosome localization, synteny analysis, conserved motif identifica-
tion, cis-acting element prediction, and expression profiling. These results provide a basis
for the subsequent exploration of the biological function of SBP genes in N. cadamba.

2. Materials and Methods

2.1. Identification of NcSBP Genes in N. cadamba
To identify the potential SBP genes in N. cadamba, the amino acid sequences of 16 SBP
genes in A. thaliana were obtained from the TAIR database (https://www.arabidopsis.
org/, accessed on 20 May 2024) and used as query sequences to search the candidate
NcSBPs via the BlastP program. Meanwhile, the hidden Markov model of the SBP gene
family domain (PF03110) was downloaded from the Pfam website (https://pfam-legacy.
xfam.org, accessed on 20 May 2024). The simple HMM Search program from TBtools
(v2.210) was used to search all the potential SBP-containing domain protein sequences of
N. cadamba. In these two ways, the candidate SBP proteins were obtained and submitted
to the NCBI Conserved Domain Search Service (CD Search) (https://www.ncbi.nlm.nih.
gov/Structure/cdd/wrpsb.cgi, accessed on 21 May 2024) to confirm their core domain
sequences. The identified NcSBP gene was then named according to the chromosome
location. The basic physicochemical properties of the NcSBP protein were analyzed with
ExPASy (http://www.expasy.org/, accessed on 23 May 2024) ProtParam. The genome
sequence and annotation information of N. cadamba was obtained from the National Center
for Biotechnology Information (NCBI) database with the accession number PRJNA650253.

2.2. Multiple Sequence Alignment and Phylogenetic Analysis

Multiple sequence alignment of the SBP domain of the NcSBP proteins was conducted
using DNAMAN software to confirm the conservation of the SBP domain. The amino acid
sequences of the A. thaliana, O. sativa, Populus trichocarpa, and N. cadamba SBP proteins were
collected for phylogenetic analysis. The accession numbers of these SBP proteins from A.
thaliana, O. sativa, and P. trichocarpa are listed in Supplementary Table S1. The phylogenetic
tree was constructed using the maximum likelihood (ML) method in the “One Step Build a
ML Tree” program from TBtools with default parameters, with 5000 bootstrap replicates.
The Interactive Tree of Life (iTOL) (https://itol.embl.de/, accessed on 26 May 2024) was
used to visualize and optimize the tree subsequently. The polygenetic tree for N. cadamba
SBP proteins was also constructed using the ML method in MEGA X with 1000 bootstrap
replicates.

2.3. Gene Structures, Conserved Motifs, and Domain Analysis

The exon–intron structures of the NcSBP genes were generated using TBtools based
on their genome DNA sequence and coding sequence (CDS). Multiple Expectation Max-
imization for Motif Elicitation (MEME) version 5.5.7 (https://meme-suite.org/meme/
index.html, accessed on 28 May 2024) was used to identify the conserved motifs of NcSBP
proteins, with the number of maximum motifs set to 10. The conserved domains of
NcSBP proteins were searched using the NCBI’s Conserved Domain Database (CDD)
(https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 28 May 2024). The
gene structures, conserved motifs, and domains were visualized with TBtools.
Genes 2025, 16, 460 4 of 20

2.4. Chromosome Localization and Collinearity Analysis

The location of NcSBPs on the chromosome was examined and mapped using the
plug-in program of Gene Location Visualize from GTF/GFF in TBtools software. To identify
the patterns of gene duplication, synteny analyses of the SBP genes in N. cadamba vs. A.
thaliana and N. cadamba vs. P. trichocarpa were conducted using the Dual Systeny Plot
program of TBtools.

2.5. Promoter cis-Acting Element Analysis

The 2 kb upstream of the transcriptional start site (ATG) of the NcSBP genes was se-
lected and considered as the gene promoter sequence. The cis-acting elements of the NcSBP
promoter were predicted using the PlantCARE online website (https://bioinformatics.
psb.ugent.be/webtools/plantcare/html/, accessed on 29 May 2024) and visualized with
TBtools.

2.6. Expression Pattern of NcSBPs

The expression patterns of the NcSBPs were analyzed using previous transcriptome
data. The different tissues, including young leaves, old leaves, bud, bark, phloem, cam-
bium, fruit, and root from 5-year-old N. cadamba were sampled for RNA-seq in a previous
study [35]. Three different types of vascular cells (cambium, phloem, and xylem) at three
developmental stages (primary growth, secondary growth, and the transition from pri-
mary to secondary growth) were isolated via laser microdissection and used for RNA
sequencing [35]. These RNA-seq data were downloaded from the NCBI under accession
number SAMN15700859. The RNA-seq data for 1-aminocyclopropane-1-carboxylic acid
(ACC, the precursor of ethylene) treatment were obtained from the Genome Sequence
Archive (https://ngdc.cncb.ac.cn/gsa/, accessed on 2 June 2024) with submission number
CRA005285 [36]. Briefly, the N. cadamba seedlings (height of 4–5 cm) were divided into two
groups: one treated with ACC (50 µmol/L, Sigma-Aldrich, St. Louis, MO, USA) and the
other with sterilized water as the control (CK). The second internode segments (counted
from the apex downward) of seedlings were collected for transcriptome sequencing after
ACC treatment for 6 h (6 h), 3 days (3 d), 7 days (7 d), and 14 days (14 d) [36]. For the auxin,
drought, and salt stress treatments, 2-month-old seedlings of N. cadamba were transferred
into MS liquid medium (MS basal salts (4.74 g/L), sucrose (30 g/L), adjusted to pH 5.8)
supplied with 100 µmol/L indole acetic acid (IAA, Sigma-Aldrich, St. Louis, MO, USA),
10% polyethylene glycol (PEG) 6000 (Macklin Biochemical, Shanghai, China) solution, and
100 mM/L NaCl (Macklin Biochemical, Shanghai, China), respectively. The leaves were
collected at 1 h, 4 h, 12 h, and 24 h after treatment. For methyl jasmonate (MeJA) (Sigma-
Aldrich, St. Louis, MO, USA) treatment, the hairy root of N. cadamba was subjected to the
MS liquid medium with 250 µmol/L MeJA and sampled after 2 h, 4 h, 8 h, 12 h, 24 h, 48 h,
72 h, and 96 h, respectively. For cold stress treatment, 3-month-old plants were transferred
to a growth chamber at 4 ◦ C, and leaves were collected after 2 h, 4 h, 8 h, 12 h, and 24 h,
respectively. Three biological replicates were collected for each sample. All samples were
immediately frozen in liquid nitrogen and kept at −80 ◦ C until RNA extraction. Total RNA
were extracted employing the E.Z.N.A.® Plant RNA Kit (Omega Bio-tek, Inc., Norcross, GA,
USA) and used for subsequent RNA-seq (data not published). The expression levels of SBP
genes in N. cadamba were quantified using fragments per kilobase of exon model per million
mapped reads (FPKM). TBtools software was used to visualize the NcSBP expression heat
map. All the FPKM values for each NcSBP in different tissues and under various treatments
are listed in Supplementary Table S2.
Quantitative real-time PCR (qRT-PCR) was used to verify the expression patterns of
the NcSBP genes. First-strand cDNA was synthesized via the reverse transcription of 1.0 µg
Genes 2025, 16, 460 5 of 20

total RNA using HiScript® III RT SuperMix for qPCR Kit (R323, Vazyme Biotech, Nanjing,
China). qRT-PCR was performed utilizing SYBR Green mix (Vazyme Biotech, Nanjing,
China) on a Roche LightCycler® 480 instrument. The cycling parameters were as follows:
95 ◦ C for 30 s, 40 cycles at 95 ◦ C for 10 s, and 60 ◦ C for 30 s. Melt-curve analyses were
performed using the following program: 95 ◦ C for 15 s, 60 ◦ C for 60 s, and 95 ◦ C for 15 s.
Three biological replicates and four technical replicates were used for each sample. The
relative expression of genes was analyzed using the 2−∆∆ct method, with the reference
gene of NcUPL (ubiquitin–protein ligase) [37]. Significance was determined by multiple
comparisons using ANOVA (p < 0.05). The gene-specific primers used in qRT-PCR are
listed in Supplementary Table S3.

3. Results
3.1. Identification and Phylogenetic Analysis of NcSBP Genes
In this study, a total of 27 SBP gene family members were identified in N. cadamba and
named as NcSBP1-NcSBP27, according to their position on chromosomes. The analysis of
the gene characteristics of the NcSBP proteins showed that the lengths of all the identified
NcSBPs ranged from 148 to 1038 amino acids. Of these, NcSBP19 was the largest protein
and NcSBP9 was the smallest protein. The molecular weight (MW) of the NcSBPs varied
from 16,714.34 to 114,331.61 Da, with the isoelectric point (pI) ranging from 5.64 to 9.76.
Interestingly, the pI of 20 NcSBPs was more than 7, while only 7 NcSBPs had a value less
than 7. The instability index of the NcSBPs ranged from 35.66 to 97.12, and the aliphatic
index was between 40.0 and 85.13. The grand average of hydropathy of the NcSBPs was
between −1.400 and −0.399, indicating that they were all hydrophilic proteins (Table 1).

Table 1. Physicochemical properties of SQUAMOSA promoter-binding proteins (SBPs) in N. cadamba.

Theoretical Grand Average

Number of Molecular Instability Aliphatic
Gene Name Isoelectric of
Amino Acids Weight (Da) Index Index
Point (pI) Hydropathicity
NcSBP1 160 18,390.48 8.99 74.72 42.12 −1.209
NcSBP2 374 41,811.45 8.62 64.18 63.16 −0.686
NcSBP3 338 35,693.41 9.17 53.40 54.05 −0.661
NcSBP4 366 39,024.07 9.19 55.27 52.27 −0.716
NcSBP5 365 40,141.66 7.16 64.17 64.16 −0.626
NcSBP6 227 25,268.04 6.48 35.66 52.47 −0.681
NcSBP7 467 50,966.04 8.66 52.99 68.37 −0.479
NcSBP8 207 23,523.51 8.47 70.94 66.91 −0.597
NcSBP9 148 16,714.34 9.47 75.61 42.97 −1.120
NcSBP10 437 47,421.41 7.96 56.03 59.89 −0.635
NcSBP11 520 57,913.78 6.86 51.09 72.02 −0.592
NcSBP12 458 504,469.66 8.92 50.94 63.67 −0.599
NcSBP13 279 31,079.96 9.76 57.12 67.78 −0.617
NcSBP14 205 22,683.08 9.24 48.41 42.39 −1.141
NcSBP15 200 23,186.48 5.64 97.12 40.0 −1.400
NcSBP16 807 89,792.49 6.55 52.81 77.41 −0.399
NcSBP17 1007 112,571.72 7.72 53.35 82.6 −0.440
NcSBP18 184 20,945.47 9.07 45.69 52.01 −1.048
NcSBP19 1038 114,331.61 6.09 49.96 85.13 −0.318
NcSBP20 259 29,184.32 9.59 67.50 38.46 −1.140
NcSBP21 1007 112,572.40 7.18 55.98 80.63 −0.440
NcSBP22 324 36,282.26 6.70 62.34 60.43 −0.694
NcSBP23 464 50,676.55 8.81 52.36 60.75 −0.630
NcSBP24 464 51,786.02 8.68 49.90 66.47 −0.642
NcSBP25 552 60,659.47 7.94 72.92 61.54 −0.808
NcSBP26 530 58,574.96 8.60 56.71 58.51 −0.677
NcSBP27 218 24,525.01 6.68 83.17 40.69 −1.258
 Genes 2025, 16, 460 6 of 22
Genes 2025, 16, 460 6 of 20

NcSBP8 207 23,523.51 8.47 70.94 66.91 −0.597

NcSBP9 To verify 148the conserved
16,714.34 9.47 75.61 42.97
SBP domain in each NcSBP protein, multiple sequence align- −1.120
NcSBP10 437 47,421.41 7.96 56.03 59.89 −0.635
ment was performed using DNAMAN software. The results showed that the SBP domains
NcSBP11 520 57,913.78 6.86 51.09 72.02 −0.592
in all NcSBP proteins exhibited conserved structural features, including two characteris-
NcSBP12 458 504,469.66 8.92 50.94 63.67 −0.599
tic zinc-finger
NcSBP13 279motifs (C3H and C2HC) and
31,079.96 9.76a bipartite nuclear
57.12 localization
67.78 signal (NLS).
−0.617
However, there
NcSBP14 205 was an 22,683.08
exception that no NLS in NcSBP6 (Figure
9.24 was found 48.41 42.39 S1). To further
−1.141
NcSBP15
investigate 200 23,186.48
the phylogenetic relationships5.64
among the SBP 97.12
proteins, 40.0 −1.400
an ML phylogenetic
NcSBP16 807
tree was generated using89,792.49
the SBP proteins 6.55 52.81 A. thaliana,
from N. cadamba, 77.41 O. sativa, and
−0.399
P.
NcSBP17 1007 112,571.72 7.72 53.35 82.6 −0.440
trichocarpa. The results displayed that the SBP proteins were divided into nine clades (clade
NcSBP18 184 20,945.47 9.07 45.69 52.01 −1.048
I to clade IX)
NcSBP19 (Figure 1),114,331.61
1038 according to the classification
6.09 established
49.96 in a85.13 on A.
previous study −0.318
thaliana
NcSBP20 and rice
259 [38], suggesting
29,184.32 that there is
9.59evolutionary conservation
67.50 between
38.46 these four
−1.140
species. The1007
NcSBP21 number of112,572.40
SBP members varied 7.18between all 55.98
nine clades.80.63
Clades II and IX−0.440
had
NcSBP22 324
the largest numbers of 36,282.26
SBP proteins, with 6.70
14 members in62.34each clade,60.43 −0.694
respectively. NcSBP
NcSBP23 464 50,676.55 8.81 52.36 60.75 −0.630
proteins were distributed in all clades except clade V, which was specific to the monocot
NcSBP24 464 51,786.02 8.68 49.90 66.47 −0.642
plant. There552
NcSBP25
was a differential
60,659.47
of NcSBP members
distribution7.94 72.92
across61.54
various clades,−0.808
with
the highest representation
NcSBP26 530 observed in clade
58,574.96 8.60IX (five members)
56.71 and the lowest in clade
58.51 III
−0.677
(one member).
NcSBP27 218 24,525.01 6.68 83.17 40.69 −1.258

Figure 1. Phylogenetic tree of SBP proteins from N. cadamba, O. sativa, P. trichocarpa, and A. thaliana.
The SBP protein sequences of 27 NcSBPs, 16 AtSBPs, 19 OsSBPs, and 29 PtSBPs were used to construct
the phylogenetic tree using the maximum likelihood (ML) method with 5000 bootstrap replicates.
The subfamilies of SBP proteins, clade I~IX, were marked with different-colored arcs. Nc: N. cadamba;
Os: O. sativa; Pt: P. trichocarpa; At: A. thaliana.
Genes 2025, 16, 460 7 of 20

3.2. Conserved Motif and Gene Structure Analysis of NcSBPs

An unrooted phylogenetic tree was constructed to align the amino acids of 27 NcSBPs
from N. cadamba (Figure 2A). The conserved motifs within the NcSBP protein sequences
were analyzed using the MEME tool. As shown in Figure 2B, motifs 1, 2, and 3 were present
in all members of the NcSBP family, indicating their potential significance in the biological
function of NcSBP. Furthermore, the types and distributions of motifs among the different
NcSBP proteins within the same clade exhibited high similarity. For instance, motifs 1, 2,
3, and 9 were consistently arranged in clade I, II, III, and IV, suggesting a high degree of
sequence similarity among these clade members. Notably, motif 9 was identified in all
members of clades I, II, III, IV, and VII, with the exception of NcSBP20. In contrast, motifs 4,
5, 6, 7, and 8 were predominantly found in NcSBP17, NcSBP19, and NcSBP21, potentially
Genes 2025, 16, 460 8 of 22
contributing to the functional specificity of these NcSBP transcription factors.

FigureFigure 2. Polygenetic
2. Polygenetic relationships, conversed
relationships, conversed motifs, and gene
motifs, andstructure of NcSBP genes.
gene structure (A) The
of NcSBP genes. (A) The
polygenetic tree was constructed based on the 27 NcSBP protein sequences using the maximum
polygenetic tree was constructed based on the 27 NcSBP protein sequences using the maximum
likelihood (ML) method with 1000 bootstrap replicates. The subfamilies of NcSBP, clade I to clade
likelihood (ML) method with 1000 bootstrap replicates. The subfamilies of NcSBP, clade I to clade
IX except clade V, were shown in different colors as the same of Figure 1. (B) Conversed motif com‐
IX except clade
position V, were
of NcSBP shown
proteins. Ten in different
motifs colors 10)
(motif 1⁓motif as are
therepresented
same of Figure 1. (B)
with different Conversed motif
colored
composition of NcSBP proteins. Ten motifs (motif 1~motif 10) are represented with
boxes. (C) Exon–intron structure of NcSBP genes. Exons and untranslated regions (UTRs) are shown different colored
boxes. in(C) Exon–intron
purple structure
and pink boxes, of NcSBP
and introns genes. Exons
are displayed and lines.
with black untranslated regions
SBP‐conserved (UTRs)
domain is are shown
shown
in purple in apink
and light green box,and
boxes, and other
intronscolors
areindicate different
displayed conserved
with blackdomains
lines. found in CDD.
SBP-conserved domain is
shown in a light green box, and other colors indicate different conserved domains found in CDD.
3.3. Chromosome Localization and Collinearity Analysis of NcSBPs
The distribution
Additionally, of NcSBP genes
the exon–intron across the chromosomes
distribution patterns of was
thepredicted
NcSBPusing
genes thewere investi-
TBtools software, based on the available gene annotation information of the N. cadamba
gatedgenome.
by comparing their coding sequences (CDS) with genomic sequences. As shown in
The results showed that the 27 NcSBP genes were unevenly distributed on 17
Figurechromosomes,
2C, we found withvariability
1‐3 NcSBP in theinnumber
genes of exons (Figure
each chromosome across 3).
different clades.
To obtain insightFor example,
members in expansion
into the clade I (NcSBP3
of the NcSBPand NcSBP4)
family, contained
we performed gene3duplication
exons, whereas three
analysis. In gen‐ members in
eral, a gene cluster is defined as a region no longer than 20 kb and containing
clade II (NcSBP2, NcSBP5, NcSBP12, and NcSBP23) exhibited 4-7 exons. The NcSBP genes two or more
genes from the same family. Accordingly, four NcSBP gene clusters (NcSBP6/NcSBP7,
within some clades shared similar exon and intron structures, such as the presence of two
NcSBP9/NcSBP10, NcSBP12/NcSBP13, and NcSBP22/NcSBP23) were characterized as tan‐
introns
deminrepeat
cladegene
I. However,
pairs, locatedcertain NcSBP chr04,
on chromosomes geneschr08,
displayed structural
chr09, and chr19. deviations from
their cladeIncounterparts.
addition to tandem instance, NcSBP14
Forduplication, and NcSBP18
fragment duplication eventsin cladetheIXNcSBP
within had significantly
gene family were also conducted. The intraspecific collinearity analysis identified 27 col‐
linear pairs among the NcSBP gene family, encompassing 26 SBP genes. Notably, no col‐
linear modules were observed on chromosomes 18 and 21 (Figure 4A). Each pair of col‐
linear genes was situated on different chromosomes and was associated with fragment
replication events. Taken together, the analysis of gene duplication events suggests that
Genes 2025, 16, 460 8 of 20

fewer exons compared to other genes in the same clade. Moreover, with the exception of
NcSBP1/6/9/20, most NcSBP genes contained at least one non-coding region (UTR). Interest-
ingly, all members of the NcSBP gene family harbored conserved SBP domains located on
two exons, which were invariably separated by an intron (Figure 2C). In addition, the SBP
domains of all the NcSBP genes, with the exception of NcSBP6, NcSBP22, and NcSBP23,
were distributed in the first and second exons, or the second and the third exons.

3.3. Chromosome Localization and Collinearity Analysis of NcSBPs

The distribution of NcSBP genes across the chromosomes was predicted using the
TBtools software, based on the available gene annotation information of the N. cadamba
genome. The results showed that the 27 NcSBP genes were unevenly distributed on 17
chromosomes, with 1-3 NcSBP genes in each chromosome (Figure 3). To obtain insight into
the expansion of the NcSBP family, we performed gene duplication analysis. In general,
a gene cluster is defined as a region no longer than 20 kb and containing two or more
genes from the same family. Accordingly, four NcSBP gene clusters (NcSBP6/NcSBP7,
NcSBP9/NcSBP10, NcSBP12/NcSBP13, and NcSBP22/NcSBP23) were characterized
Genes 2025, 16, 460 10 of 22
as
tandem repeat gene pairs, located on chromosomes chr04, chr08, chr09, and chr19.

Figure 3. Chromosome location

Figure of NcSBP
3. Chromosome genes.
location The
of NcSBP scale
genes. Thebar
scaleon thetheleft
bar on indicates
left indicates the chromosome
the chromosome

length. Gene densitieslength. Gene densities are drawn based on the annotation data of the N. cadamba genome, with red
are drawn based on the annotation data of the N. cadamba genome, with red
representing high density and blue representing low density. Red font size markers are gene names,
representing high density and
and chr bluechromosome.
indicates representing low density. Red font size markers are gene names,
and chr indicates chromosome.
Genes 2025, 16, 460 9 of 20

In addition to tandem duplication, fragment duplication events within the NcSBP gene
family were also conducted. The intraspecific collinearity analysis identified 27 collinear
pairs among the NcSBP gene family, encompassing 26 SBP genes. Notably, no collinear
modules were observed on chromosomes 18 and 21 (Figure 4A). Each pair of collinear
genes was situated on different chromosomes and was associated with fragment replication
events. Taken together, the analysis of gene duplication events suggests that fragment
Genes 2025, 16, 460 11 of 22
replication serves as the main driving force behind the expansion of the SBP gene family.

Figure 4. Synteny analysis of SBP genes within N. cadamba (A) and between N. cadamba and two
representative species of A.Figure
thaliana and P. trichocarpa (B) Gray lines in the background represent the
4. Synteny analysis of SBP genes within N. cadamba (A) and between N. cadamba and two
synteny blocks in the genomes, and black
representative or of
species red
A. lines
thalianaindicate duplication
and P. trichocarpa SBP
(B) Gray gene
lines in thepairs.
background represent the
synteny blocks in the genomes, and black or red lines indicate duplication SBP gene pairs.
To further elucidate the phylogenetic mechanisms of the N. cadamba SBP family, com-
parative syntenic maps were constructed, integrating N. cadamba with two representative
species of A. thaliana and P. trichocarpa (Figure 4B). The results revealed 22 SBP orthologous
gene pairs between N. cadamba and A. thaliana. They were identified between 11 chromo-
somes of N. cadamba and 4 chromosomes of A. thaliana. Notably, the collinearity blocks were
predominantly concentrated in chromosome At-1 of A. thaliana. Additionally, 55 collinear
gene pairs were identified between N. cadamba and P. trichocarpa. Many collinearity blocks
were observed between chr15 of N. cadamba and chromosome Pt-14 of P. trichocarpa. How-
ever, no synteny blocks were found in N. cadamba chromosomes Nc-06, 07, 11, 15, 16, or 21,
Genes 2025, 16, 460 10 of 20

or in P. trichocarpa chromosomes Pt-6, 9, 13, or 17. These findings suggest that NcSBP genes
exhibit a closer phylogenetic relationship with the SBP genes of P. trichocarpa compared to
those of A. thaliana. Furthermore, certain NcSBP genes, such as NcSBP27, share multiple
orthologous gene pairs with both P. trichocarpa and A. thaliana. Conversely, some genes
display collinearity predominantly with one species. For instance, NcSBP8 on chr05 of N.
cadamba shares three orthologous gene pairs with P. trichocarpa but none with A. thaliana,
suggesting its potential role in the growth and development of woody plants specifically.

3.4. cis-Acting Element Analysis of NcSBP Promoters

To enhance our understanding of transcriptional regulation and the possible roles
of NcSBPs in N. cadamba, we predicted the cis-acting elements present in the promoters
of NcSBP genes using Plant CARE (Figure 5A). The cis-acting elements identified in the
promoter regions of NcSBP genes were categorized into four groups, including stress re-
sponse elements, hormone response elements, light response elements, and plant growth
and development response elements. Among these, hormone response elements and light
response elements were the most abundant. Specifically, the promoters of NcSBP1, NcSBP5,
NcSBP10, NcSBP17, NcSBP19, NcSBP20, and NcSBP26 contained 12 or more hormone
response cis-acting elements, suggesting their potential significance in hormone-mediated
regulatory processes. Within the hormone response elements, ABRE (abscisic acid response
element), TGACG motif (MeJA response element), and AuxRR core (auxin response ele-
ment) were particularly prevalent. In terms of light response elements, the GATA motif
was notably abundant, with NcSBP14, NcSBP21, and NcSBP26 each containing six of such
elements. Additionally, stress-related elements, including the drought-responsive MYB
binding site (MBS) and the low-temperature response element (LTR), were widely dis-
tributed, appearing in over 70% of the NcSBP promoter regions (Figure 5B). These findings
Genes 2025, 16, 460 highlight the diverse regulatory roles of NcSBP genes in responding 12 toofenvironmental
22

stresses, hormonal signals, and light conditions.

5. Cis-acting
FigureFigure element
5. Cis‐acting analysis
element analysis of the
of the promoter
promoter region
region of NcSBPof NcSBP
genes. genes.
(A) The (A) The
distribution of distribution
cis‐acting elements in NcSBP promoters. Blocks with different colors represent various
of cis-acting elements in NcSBP promoters. Blocks with different colors represent various types types of cis‐
acting elements.
of cis-acting elements. (B) The
(B)number of cis‐acting
The number ofelements related
cis-acting to stress response,
elements related hormone
to stressresponse,
response, hormone
light response, and growth and development in NcSBP promoters.
response, light response, and growth and development in NcSBP promoters.
3.5. Expression Patterns of NcSBP Genes in Various Tissues
3.5. Expression Patterns of NcSBP Genes in Various Tissues
To investigate the possible role of NcSBP in the development of various tissues and
To investigate
organs, the the
we analyzed possible
expression of NcSBP
rolepatterns of 27 in the genes
NcSBP development of various
across different tissues, tissues and
organs, we analyzed
including bud, bark,the expression
young leaves, old patterns
leaves, root, 27 NcSBP
ofyoung genes across
fruit, cambium, different tissues,
and phloem,
utilizing
including previously
bud, acquiredleaves,
bark, young transcriptome data (Figure
old leaves, 6).young
root, The results showed
fruit, that mostand phloem,
cambium,
NcSBP genes were expressed in diverse tissues. Notably, NcSBP3 and NcSBP9 exhibited
high expression levels in buds, whereas NcSBP1 demonstrated the highest expression in
young leaves. NcSBP6 was predominantly expressed in bark and young fruit, but
NcSBP14 showed elevated expression in cambium and phloem, while NcSBP20 was spe‐
cifically expressed in old leaves and roots (Figure 6A). To examine the expression profiles
of NcSBP in various developmental vascular tissues, we employed laser microdissection
Genes 2025, 16, 460 11 of 20

utilizing previously acquired transcriptome data (Figure 6). The results showed that most
NcSBP genes were expressed in diverse tissues. Notably, NcSBP3 and NcSBP9 exhibited
high expression levels in buds, whereas NcSBP1 demonstrated the highest expression in
young leaves. NcSBP6 was predominantly expressed in bark and young fruit, but NcSBP14
showed elevated expression in cambium and phloem, while NcSBP20 was specifically
expressed in old leaves and roots (Figure 6A). To examine the expression profiles of NcSBP
in various developmental vascular tissues, we employed laser microdissection to isolate
cambium, phloem, and xylem cells at three distinct stages: primary growth, secondary
growth, and the transitional stage from primary to secondary growth [39]. Following
this, RNA sequencing was performed. The expression profiling analysis revealed distinct
spatial–temporal
Genes 2025, 16, 460
expression patterns of NcSBP genes during vascular development 13 of 22
in
N. cadamba. Obviously, NcSBP17 exhibited consistently high expression levels across the
three developmental stages in all vascular tissues (cambium, phloem, and xylem), except
To further validate the expression profiles of NcSBPs across various tissue types,
in cambium cells at the transition
qRT‐PCR stage,tosuggesting
was employed its significant
assess the expression levels of fiverole
NcSBPsin (NcSBP3,
vascular tissue
NcSBP6,
differentiation and development in N.
NcSBP9, NcSBP14, cadamba.
and that exhibited highNcSBP23
NcSBP20) Additionally, expression had hightissues
in different expression
(Fig‐
ure 6C–G). Consistent with the transcriptome data, the qRT‐PCR analysis revealed that
during the primary growth and transition stage of phloem development, and NcSBP14
NcSBP3 and NcSBP9 were most highly expressed in buds and young leaves, while NcSBP6
was specifically highlyshowed
expressed during the
strong expression transition
in fruits stage
(Figure 6C–E). of vascular
However, cambium
discrepancies growth.
were found in
Furthermore, NcSBP15theshowed
expressionhigh expression
patterns of NcSBP14inand theNcSBP20
primary xylem,
compared while
to the NcSBP22
transcriptome was
data.
Specifically, NcSBP14 was predominantly expressed in fruits (Figure 6F), whereas the
predominantly expressed in the phloem and cambium during secondary growth. These
transcriptome analysis indicated high expression in the phloem and cambium (Figure 6A).
differential expressionAdditionally,
patterns strongly suggest that
NcSBP20 demonstrated NcSBP
high genes
expression functionally
in various specialized
tissues, excluding old
leaves and roots (Figure 6G). It is hypothesized that the differences
in regulating specific stages of vascular tissue development and differentiation processes in in expression patterns
may be attributed to variations in the sample sources utilized for transcriptome analysis
N. cadamba. and qRT‐PCR.

Figure of
Figure 6. Expression patterns 6. Expression
NcSBPs patterns of NcSBPs
in different in different
tissues and tissues
vascularand vascular
cells atcells at three
three developmen‐
developmental
tal stages. Expression of NcSBP genes in various tissues. (A) YL, young leaves; B, bark; C, cambium;
stages. Expression of NcSBP genes in various tissues. (A) YL, young leaves; B, bark; C, cambium;
OL, old leaves; FR, fruit; R, root. (B) NcSBPs transcript levels in cambium, phloem, and xylem cells
OL, old leaves; FR, fruit;atR,different (B) NcSBPsstages.
root. developmental transcript levels
PX, primary in cells;
xylem cambium,
TX, xylemphloem,
cells at theand xylem
transitional cells
stage
at different developmentalfromstages.
primary PX, primary
to secondary xylem
growth; cells; TX,xylem
SX, secondary xylem cells;cells
TCA, at the transitional
cambium stage
cells at the transi‐
from primary to secondary tional stage from
growth; SX,primary to secondary
secondary xylemgrowth;
cells; SCA,
TCA,secondary
cambium cambium
cells cells;
at thePPH, primary
transitional
phloem cells; TPH, phloem cells at the transitional stage from primary to secondary growth; SPH,
stage from primary to secondary growth; SCA, secondary cambium cells; PPH, primary phloem
secondary phloem cells. Heatmaps were generated using TBtools, utilizing transformed log2
cells; TPH, phloem cells at the transitional
(FPKM+1) stageanalysis
values, and cluster from primary to secondary
was conducted growth;levels
on the gene expression SPH,bysecondary
row. The
phloem cells. Heatmaps color
werebar generated using
indicated gene TBtools,
expression, withutilizing transformed
red representing log2and
high expression (FPKM+1) values,
blue representing
Genes 2025, 16, 460 12 of 20

and cluster analysis was conducted on the gene expression levels by row. The color bar indicated
gene expression, with red representing high expression and blue representing low expression. (C–G)
Gene expression analysis of 5 selected NcSBP genes in eight tissues of N. cadamba by qTR-PCR. NcUPL
was used as the reference gene, and transcript levels in old leaves were set as the calibrator (assigned
a value of 1). Relative expression in other tissues was then determined accordingly. Error bars
represent standard deviations of mean value from three biological replicates. Different letters indicate
statistically significant differences between groups based on ANOVA (p < 0.05).

To further validate the expression profiles of NcSBPs across various tissue types, qRT-
PCR was employed to assess the expression levels of five NcSBPs (NcSBP3, NcSBP6, NcSBP9,
NcSBP14, and NcSBP20) that exhibited high expression in different tissues (Figure 6C–G).
Consistent with the transcriptome data, the qRT-PCR analysis revealed that NcSBP3 and
NcSBP9 were most highly expressed in buds and young leaves, while NcSBP6 showed
strong expression in fruits (Figure 6C–E). However, discrepancies were found in the expres-
sion patterns of NcSBP14 and NcSBP20 compared to the transcriptome data. Specifically,
NcSBP14 was predominantly expressed in fruits (Figure 6F), whereas the transcriptome
analysis indicated high expression in the phloem and cambium (Figure 6A). Additionally,
NcSBP20 demonstrated high expression in various tissues, excluding old leaves and roots
(Figure 6G). It is hypothesized that the differences in expression patterns may be attributed
to variations in the sample sources utilized for transcriptome analysis and qRT-PCR.

3.6. Expression Analysis of NcSBPs in Response to Hormones and Abiotic Stress Treatment
To investigate whether the NcSBP genes were response to different hormones, ex-
pression patterns of all the identified NSBPs were analyzed using the RNA-seq data. The
results demonstrated that ACC treatment significantly up-regulated the expression levels
of NcSBP14 after 1 day of treatment compared to the control group. Notably, NcSBP8 and
NcSBP26 exhibited the most pronounced up-regulation on day 3 post-treatment (Figure 7A).
As for MeJA treatment, the expression of NcSBP2, NcSBP20, NcSBP10, and NcSBP26 was
up-regulated at 2 h after treatment initiation. The expression of NcSBP7 peaked at 4 h,
while NcSBP6/9/15 reached their highest levels under MeJA treatment at 8 h. Con-
versely, NcSBP13 and NcSBP17 showed down-regulated expression after 72 h and 96 h
of treatment, respectively (Figure 7B). Under IAA treatment, the expression levels of
NcSBP1/2/4/5/12/15/23/25 gradually decreased with prolonged treatment time. In con-
trast, NcSBP8 expression was induced and peaked at 4 h, while NcSBP11/14/19/20/25 and
NcSBP13/17 reached their highest expression levels at 1 h and 12 h, respectively (Figure 7C).
These findings indicate that the expression patterns of NcSBP genes varied under ACC,
MeJA, and IAA treatments, suggesting that NcSBPs may play distinct roles in different
hormone response pathways.
To elucidate the response mechanisms of NcSBP genes to abiotic stress, we analyzed
transcriptome data under various stress treatments, including low temperature (4 ◦ C),
drought (stimulated by PEG), and salt (NaCl) (Figure 8). Under low-temperature stress, the
expression levels of NcSBP2/7/10/11/14/19/21/23/24/25 decreased gradually with prolonged
treatment. In contrast, NcSBP3/5/9/12/16 reached the highest transcript levels at 2 h post-
treatment before declining. Notably, NcSBP6, NcSBP8, and NcSBP13 exhibited delayed
responses, reaching their highest expression levels after 24 h of treatment, suggesting a
slower adaptation to low-temperature stress. For PEG treatment, the expression levels
of NcSBP1/2/4/5/12/15/18/21/26/27 declined over time. Conversely, NcSBP11, NcSBP19,
NcSBP20, and NcSBP25 peaked at 2 h before decreasing. Meanwhile, NcSBP6/13/22/24
and NcSBP10 reached their highest expression levels at 12 h and 24 h, respectively, with
low expression at other time points. Under NaCl conditions, the expression levels of
NcSBP1/6/13/16/20/23 were significantly down-regulated. In contrast, NcSBP2/7/9/10/11
 7A). As for MeJA treatment, the expression of NcSBP2, NcSBP20, NcSBP10, and NcSBP26
was up‐regulated at 2 h after treatment initiation. The expression of NcSBP7 peaked at 4
Genes 2025, 16, 460 h, while NcSBP6/9/15 reached their highest levels under MeJA treatment at 8 h. Con‐
13 of 20
versely, NcSBP13 and NcSBP17 showed down‐regulated expression after 72 h and 96 h of
treatment, respectively (Figure 7B). Under IAA treatment, the expression levels of
NcSBP1/2/4/5/12/15/23/25 gradually decreased with prolonged treatment time. In con‐
exhibited an initial increase at 1 h, followed by a gradual decline. NcSBP3/5/14/19/21/25 had
trast, NcSBP8 expression was induced and peaked at 4 h, while NcSBP11/14/19/20/25 and
the highest expression at 4 h, while NcSBP15 and NcSBP27 showed elevated expression
NcSBP13/17 reached their highest expression levels at 1 h and 12 h, respectively (Figure
only at 24 h, 7C).
remaining low at other
These findings time
indicate thatpoints. Taken together,
the expression patterns ofthese
NcSBPresults
geneshighlight
varied under
the diverse and dynamic expression patterns of NcSBP genes under different
ACC, MeJA, and IAA treatments, suggesting that NcSBPs may play distinct abioticroles
stress
in dif‐
conditions, suggesting theirresponse
ferent hormone involvement in distinct stress response mechanisms.
pathways.

Genes 2025, 16, 460 15 of 22

the expression levels of NcSBP2/7/10/11/14/19/21/23/24/25 decreased gradually with pro‐

longed treatment. In contrast, NcSBP3/5/9/12/16 reached the highest transcript levels at 2
h post‐treatment before declining. Notably, NcSBP6, NcSBP8, and NcSBP13 exhibited de‐
layed responses, reaching their highest expression levels after 24 h of treatment, suggest‐
ing a slower adaptation to low‐temperature stress. For PEG treatment, the expression lev‐
els of NcSBP1/2/4/5/12/15/18/21/26/27 declined over time. Conversely, NcSBP11, NcSBP19,
NcSBP20, and NcSBP25 peaked at 2 h before decreasing. Meanwhile, NcSBP6/13/22/24 and
NcSBP10
Figure
Figure 7. Expression 7. reached
Expression
pattern their highest
pattern
of NcSBPs atofdifferent
NcSBPs expression levels
at different
times under at under
times 12 hhormone
various and 24 h,treatments.
various respectively,
hormone (A)with
treatments. low
Ex- (A)
expression
pression levelsExpression
of NcSBPslevels at of
under other
NcSBPs
ACC timeunderpoints. Under NaCl conditions,
ACC (1‐aminocyclopropane‐1‐carboxylic
(1-aminocyclopropane-1-carboxylic the expression
acid, precursoracid, of
precursor levels
of eth‐of
ethylene,
NcSBP1/6/13/16/20/23
ylene,
50 µmol/L) treatment 50 and
μmol/L) treatment
the sterilized were
and
water significantly
the down‐regulated.
sterilized water
treatment (CK). treatment (CK).In
(B) Expression (B) contrast,
Expression
levels NcSBP2/7/9/10/11
levelsunder
of NcSBPs of NcSBPs
exhibited
under MeJA an initial
(250 increase
μmol/L) at
treatment. 1 h,
(C) followed
Expression by a gradual
analysis of
MeJA (250 µmol/L) treatment. (C) Expression analysis of NcSBPs under IAA (100 µmol/L) treatment. decline.
NcSBPs NcSBP3/5/14/19/21/25
under IAA (100 μmol/L)
had the
treatment. highest
The expression
heatmaps were at 4
generated h, while
using NcSBP15
TBtools and
based
The heatmaps were generated using TBtools based on the transcriptome data, with transformed NcSBP27
on the showed
transcriptome elevated
data, withexpres‐
trans‐
sion only
formed data
data of log2 (FPKM+1) at 24 h, remaining
of logGene
values. 2(FPKM+1) low at other
values.clustering
expression time
Gene expression points. Taken together,
clustering row-wise.
was performed was performed these results
bar high‐
row‐wise.
The color The
light
indicated genecolor the
bar
expression diverse
indicated and
level,gene
with dynamic
expression expression patterns
level, with high
red representing red of NcSBP
representing
expression high genes
andexpression underanddifferent
blue representing abiotic
blue represent‐
low
stress
expression. d: ing
day,low conditions,
h: expression.
hour. d:suggesting
day, h: hour. their involvement in distinct stress response mechanisms.

To elucidate the response mechanisms of NcSBP genes to abiotic stress, we analyzed

transcriptome data under various stress treatments, including low temperature (4 °C),
drought (stimulated by PEG), and salt (NaCl) (Figure 8). Under low‐temperature stress,

Figure 8. Expression
Figure 8.pattern of NcSBPs
Expression at NcSBPs
pattern of different times under
at different times cold
under(A),
colddrought (B), (B),
(A), drought andand
saltsalt (C)
(C) stress. The stress.
seedlings of N. cadamba were treated with low temperature (4 ◦ C), 10% PEG6000,
The seedlings of N. cadamba were treated with low temperature (4 °C), 10% PEG6000, and
and NaCl (100 NaCl
mM/L), respectively.
(100 mM/L), The heatmaps
respectively. The heatmaps wereweregenerated
generatedusing TBtoolsbased
using TBtools basedon on
the the
transcrip‐
transcriptome data,
tomewith
data,transformed data of
with transformed log2(FPKM+1)
data of log2(FPKM+1) values. Gene
values. expression
Gene expressionclustering
clusteringwas
was per‐
performed row-wise.
formedThe color bar
row‐wise. Theindicates
color bargene expression,
indicates with redwith
gene expression, representing high expression
red representing high expression
and blue representing
and bluelow expression.
representing lowh:expression.
hour. h: hour.

To confirm theToexpression
confirm the patterns NcSBPsofinNcSBPs
ofpatterns
expression response to hormone
in response and abiotic
to hormone and abiotic
stresses, qRT‐PCR was employed to assess the expression levels of six
stresses, qRT-PCR was employed to assess the expression levels of six selected NcSBPs selected NcSBPs
(NcSBP7, NcSBP11, NcSBP14, NcSBP20, NcSBP22, and NcSBP25)
(NcSBP7, NcSBP11, NcSBP14, NcSBP20, NcSBP22, and NcSBP25) under IAA, PEG6000, and under IAA, PEG6000,
and NaCl treatment (Figure 9). The results showed that NcSBP11, NcSBP14, NcSBP20, and
NaCl treatment (Figure 9). The results showed that NcSBP11, NcSBP14, NcSBP20, and
NcSBP25 reached the highest expression level after 1 h under IAA and PEG treatment
NcSBP25 reached the highest expression level after 1 h under IAA and PEG treatment
(Figure 9A,B), indicating a rapid response to these two treatments. On the contrary,
NcSBP7 exhibited delayed responses, reaching their highest expression levels at 24 h post‐
treatment. Notably, NcSBP22 had the highest expression at 4 h under IAA treatment,
whereas it exhibited the highest expression at 12 h under PEG treatment. Under the NaCl
condition, NcSBP7, NcSBP11, NcSBP20, and NcSBP25 reached their highest expression
quantity at 1 h post‐treatment (Figure 9C), indicating a rapid response to NaCl. The dif‐
Genes 2025, 16, 460 14 of 20

(Figure 9A,B), indicating a rapid response to these two treatments. On the contrary, NcSBP7
exhibited delayed responses, reaching their highest expression levels at 24 h post-treatment.
Notably, NcSBP22 had the highest expression at 4 h under IAA treatment, whereas it
exhibited the highest expression at 12 h under PEG treatment. Under the NaCl condition,
NcSBP7, NcSBP11, NcSBP20, and NcSBP25 reached their highest expression quantity at
1 h post-treatment (Figure 9C), indicating a rapid response to NaCl. The difference is that
NcSBP7 and NcSBP11 remained low at other time points, while NcSBP20 and NcSBP25 had
low expression only at 12 h. It is worth noting that NcSBP14 reached the highest expression
Genes 2025, 16, 460 16 of 22
level at 4 h, while NcSBP22 increased gradually with prolonged treatment. All together,
these results suggest that the expression patterns of these NcSBPs detected by qRT-PCR
treatment. All together, these results suggest that the expression patterns of these NcSBPs
were consistent with the transcriptome data.
detected by qRT‐PCR were consistent with the transcriptome data.

Figure 9. Analysis of 6 selected NcSBPs at different times under IAA (A), PEG (B), and NaCl (C)
treatments by qRT-PCR. NcUPL served as the reference gene, with transcript levels before treatment
(0 h) normalized to 1 for relative quantification in other treatments. Error bars represent standard
deviations of mean value from three biological replicates. Groups marked with different letters differ
significantly (ANOVA, p < 0.05).
Genes 2025, 16, 460 15 of 20

4. Discussion
The SBP family is a significant transcription factor family exclusive to plants, recog-
nized for their role in regulating flower and fruit development, as well as various other
essential physiological processes. With the advancement of next-generation sequencing
technologies, SBP gene families have been widely identified in numerous species. However,
there is no research on SBP genes in N. cadamba, an important timber tree with high medici-
nal value in subtropical Asian regions. In the present study, we systematically identified
and characterized the SBP gene family in N. cadamba and performed a comprehensive
analysis with regard to phylogenetic relationships, protein properties, gene structure, chro-
mosome localization, collinearity, cis-acting elements in promoters, expression patterns in
different tissues, and responses to various hormone and abiotic stresses.
In general, the SBP gene family is relatively small in terms of transcription factors in
plants, with the majority comprising fewer than 40 members. For instance, there are 16 mem-
bers in A. thaliana [38], 19 members in rice [38], 15 members in sweet orange (Citrus sinen-
sis) [40], and 32 members in blueberry (Vaccinium uliginosum L.) [41]. In our study, a total of
27 SBP genes were identified, which was the same as apple (Malus × domestica Borkh) [8].
The number of SBP genes differed among the various plant species; however, this variation
did not correspond proportionally with alterations in genome size [42]. The tea plant (Camel-
lia sinensis) genome size (3.14 Gb and 3.02 Gb) was much greater than N. cadamba (744.5 Mb),
but only 20 SBP members were identified in tea plant. The variation in the number of
SBP genes across different plant species may be due to gene duplication or the prolonged
expansion of certain LTR retrotransposon families [43,44]. Generally, gene duplication
events, including segmental and tandem duplications, are significant contributors to the
emergence of new genes and the expansion of gene families, facilitating the adaptation of or-
ganisms to diverse and complex environments. To date, tandem duplication and segmental
duplication have been extensively characterized within the SBP gene family. In our study,
we identified four gene clusters (NcSBP6/NcSBP7, NcSBP9/NcSBP10, NcSBP12/NcSBP13,
and NcSBP22/NcSBP23), which were classified as tandem duplicate pairs in N. cadamba.
Additionally, two tandem duplicate pairs of 16 SBP genes in A. thaliana were located in the
segmental repeat region [38]. In perennial plants, 11 pairs of 29 SBP genes in P. trichocarpa
arose from intrachromosomal duplication [44], while 27 inter-chromosomal segmental
duplication events were identified among 28 EjSBP genes in loquat (Eriobotrya japonica) [45].
In our study, we identified 27 pairs of genes situated within duplicated genomic regions
in N. cadamba (Figure 3). These findings suggest that the duplication in SBP gene family
members is widespread and relatively conserved across the plant.
Gene structure analysis revealed that NcSBP genes exhibited a range of 1 to 11 exons,
likely resulting from the evolutionary processes of intron and exon insertion and deletion
within NcSBPs. However, the majority of genes consisted of three to four exons, suggesting
that NcSBPs exhibit a relatively conserved structure. According to the phylogenetic tree,
27 NcSBPs were categorized into eight distinct subfamilies, which was similar to the
phylogenetic structure observed in AtSPLs [38]. Members of A. thaliana and N. cadamba
were found across various subfamilies, with NcSBPs within the same subfamily exhibiting
comparable motifs and structural features (Figure 2). This suggests that these genes may
have originated from a common ancestor and might serve analogous roles in plant growth
and development. Furthermore, the majority of NcSBPs exhibited a closer clustering with
SBP genes from A. thaliana and P. trichocarpa, rather than those from rice (Figure 1). This
finding is consistent with the knowledge that A. thaliana and P. trichocarpa are eudicots,
which separated more recently from a common ancestor than the lineage that gave rise to
monocots. Comparative genomic analysis provides a powerful approach for extrapolating
genomic insights gained from one taxon that has been extensively investigated in terms of
Genes 2025, 16, 460 16 of 20

its genome structure, biological function, and evolutionary dynamics to less-studied species.
Consequently, the putative functions and regulatory mechanism of SBP genes in N. cadamba
can be inferred by comparing them with their orthologous genes in well-characterized
model plants like A. thaliana and P. trichocarpa. In this study, the synteny analysis of the
duplicated blocks between the N. cadamba genome and A. thaliana genome indicated that
22 pairs of SBP genes were located in syntenic genomic regions, containing 13 AtSBP genes
and 17 NcSBP genes (Figure 4B). To date, the majority of AtSBP genes, including AtSPL2,
AtSPL3, AtSPL4, AtSPL5, AtSPL7, AtSPL8, AtSPL9, AtSPL10, AtSPL11, AtSPL12, AtSPL13,
AtSPL14, and AtSPL15, have been well functionally characterized [15,46–50]. Therefore, the
possible functions of the NcSBP homologs can be deduced from their analogous proteins,
and further experimental studies are necessary to confirm these predictions.
The SBP family serve as crucial regulators of various biological and physiological
processes in plants. Expression pattern analysis provides important clues for exploring
the function of SBP genes in non-model plants. NcSBP genes exhibited wide expression
across various tissues we tested, with NcSBP1, NcSBP3, and NcSBP14 demonstrating high
expression levels in most tissues (Figure 6), implying their critical importance. Accumulat-
ing evidence indicates that SBP genes are involved in the regulation of fruit development.
For instance, SPL genes are expressed at higher levels in flower buds and young fruits in
Prunus mume [51]. SlSPL-CNR in tomato is predominantly expressed during the ripening of
fruits and plays a crucial role in promoting fruit ripening and regulating cell death [52]. In
our study, NcSBP6 exhibited extremely high expression in the fruit of N. cadamba, whereas
the low transcript levels in other NcSBPs (Figure 6A,D) imply a potential regulatory role
in fruit development. Given the importance of wood formation in perennial trees, we
concentrated our analysis on the expression patterns observed in the xylem, cambium,
and phloem during different developmental stages. Some NcSBP genes have high expres-
sion levels with high overlapping. For example, NcSBP17 displayed high expression in
all vascular cells at the three various stages, except in cambium at the transition stage
(Figure 6B), indicating its significant role in regulating vascular cell development. TaSPL14
from bread wheat has been identified as a crucial regulator in various vascular cell types in
root, including protoxylem, protophloem, and companion cells [53]. However, information
about the regulation of SBP genes in vascular tissues, especially in woody plants, is still
largely unknown. Further studies are needed to investigate the specific functions of NcSBP
on the wood formation of N. cadamba.
SBP genes have been characterized to play diverse, significant roles in response to
various hormones and stresses. The expression of SBP genes was affected by different
hormone and stress treatments. The expression of AtSPL9 decreased under NaCl and
drought treatment, and increased following recovery from these stress conditions [54],
similarly to TaSPL6 from wheat under high temperature, dehydration, and ABA stress [55].
Promoters of most NcSBP genes contain abundant response elements associated with
hormones and stress (Figure 5B), suggesting their potential involvement in regulatory
processes mediated by hormones and stress. NcSBP14/19/20/25 were significantly increased
under IAA treatment, consistent with the fact that they contained abundant auxin response
element (AuxRR). Likewise, NcSBP19/20/24 have a drought-responsive MYB binding site
(MBS) element, and they were found to be up-regulated under PEG stress conditions.
These results revealed consistency between the cis-acting regulatory elements and the
expression patterns in response to hormone or stress treatments. Previous studies have
revealed that SBPs are important regulators in linking hormone signaling in response to
environmental stresses. VvSBP8/13 from grape, targeted by miR156, function downstream
of the ABA signaling pathway to modulate anthocyanin biosynthesis in grapevine fruit
during drought conditions [56]. CaSBP13 acts as a negative regulator of drought tolerance
Genes 2025, 16, 460 17 of 20

in pepper (Capsicum annuum), likely through the modulation of ROS and ABA signaling
pathways [57]. In our study, certain NcSBP genes were activated by both hormonal signals
and abiotic stressors. Specifically, NcSBP20 and NcSBP25 were responsive to IAA and MeJA,
as well as to drought and low-temperature stress. The expression of NcSBP10 was elevated
in response to IAA, MeJA, PEG, and NaCl treatments (Figures 7 and 8). In apple, many
MdSBP genes exhibited either up-regulation or down-regulation in response to various
plant hormones, including ethylene, salicylic acid (SA), MeJA, ABA, and GA [8]. In contrast,
several SBP genes from tea plant, including CsSBP5, CsSBP15, CsSBP16, and CsSBP19, were
repressed under cold, drought, ABA, GA, and MeJA treatments [43]. These results indicate
that the SBP gene family may play a significant role in the interplay between various plant
hormones and environmental stresses.

5. Conclusions
In summary, a total of 27 NcSBP genes were identified in N. cadamba and classified
into eight clades according to phylogenetic analysis. Their gene structures, conserved
motifs, and collinearity were also investigated. The expression profile of NcSBPs across
various tissues indicated their potential roles in the growth and development of N. cadamba.
Furthermore, the analysis of cis-acting elements and expression patterns of NcSBP genes
highlighted their significant involvement in regulating responses to different hormones
and abiotic stresses. Our study lays a robust foundation for further investigation into the
SBP-mediated molecular mechanisms underlying physiological developmental processes
and stress responses.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/genes16040460/s1, Figure S1: Alignment of SBP domains of
NcSBP proteins; Table S1: The accession numbers of the SBP proteins used for ML phylogenetic tree
construction from different species; Table S2: Expressions of NcSBP gene from transcriptomic data;
Table S3: The specific primers used in the qRT-PCR.

Author Contributions: Conceptualization, J.L.; methodology, L.T. and K.L.; validation, C.C.; formal
analysis, W.W.; investigation, G.J.; resources, C.P.; writing-original draft preparation, L.T.; writing-
review and editing, Z.L.; visualization, C.C. and K.L.; funding acquisition, J.L. All authors have read
and agreed to the published version of the manuscript.

Funding: This research was financially supported by Guangdong Basic and Applied Basic Research
Foundation (2023A1515030250, 2021A1515010816) and the National Natural Science Foundation of
China (32271908, 31800560).

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The data presented in this study are available in the Supplementary
Materials.

Conflicts of Interest: The authors declare no conflicts of interest.

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