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Article

Genome-Wide Identification and Expression Profiling of Phosphatidylethanolamine-Binding Protein (PEBP) Genes in Helianthus annuus L.

by
Yiyi Sun
1,
Yanwen Wang
1,
Jingyan Bai
1,
Jiatong Guo
1,
Guiting Li
2,3,4,
Qiuzhen Tian
2,3,4,
Shuping Lv
2,5,
Hengchun Cao
2,3,4,
Xiaojie Yang
2,5,* and
Lingyun Liu
1,*
1
School of Life Sciences, Henan University, Kaifeng 475001, China
2
Henan Key Laboratory of Specific Crops Genomics, Henan Sesame Research Center, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
3
Henan Joint Key Laboratory of Specific Oilseed Crops, Zhengzhou 450002, China
4
Henan Sesame Research Center, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
5
Economic Crop Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(10), 4602; https://doi.org/10.3390/ijms26104602
Submission received: 6 March 2025 / Revised: 28 April 2025 / Accepted: 8 May 2025 / Published: 11 May 2025
(This article belongs to the Section Molecular Genetics and Genomics)
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Abstract

:
The phosphatidylethanolamine-binding protein (PEBP) gene family is critical for regulating plant growth, development, and flowering. Sunflower (Helianthus annuus L.) is the fourth most important oilseed crop globally. However, the genomic structure and functional diversity of PEBP genes in sunflower remain unexplored. Leveraging the recently assembled telomere-to-telomere (T2T) sunflower genome, a genome-wide analysis of the HaPEBP family was carried out. A total of 12 PEBP genes were identified in sunflower and categorized into three subfamilies: TFL1-like, FT-like, and MFT-like. Phylogenetic and synteny analyses revealed that tandem duplication events have substantially contributed to the evolution and expansion of the HaPEBP gene family. Furthermore, the analysis of the promoter regions revealed 77 distinct cis-acting elements, including 35 related to light signaling and growth regulation, highlighting their potential involvement in the regulation of flowering and development in sunflower. Expression profile analysis using RNA-seq data across various tissues indicated that FT-like and TFL1-like HaPEBP genes may be the key regulators of flowering time and plant architecture in sunflower varieties. This study offers valuable insights into the structural, evolutional, and functional dynamics of the HaPEBP gene family and holds significant implications for sunflower breeding strategies aimed at optimizing flowering time and plant architecture traits.

1. Introduction

The phosphatidylethanolamine-binding protein (PEBP) gene family represents an ancient and highly conserved group of genes present across all eukaryotic kingdoms, including bacteria, animals, and plants [1,2]. In plants, PEBP genes serve as key regulators of diverse developmental processes, including flowering time, plant architecture, and responses to environmental stress [3]. These genes encode proteins that participate in multiple signaling pathways essential for plant growth and differentiation. Although the functions of specific PEBP genes have been widely studied, comprehensive analyses of the entire PEBP gene family have been carried out in only a limited number of species, such as Arabidopsis, soybean, rice, tomato, barley, and pepper [4,5,6,7,8].
The PEBP gene family comprises three main subfamilies: FLOWERING LOCUS T (FT)-like, TERMINAL FLOWER1 (TFL1)-like, and MOTHER OF FT AND TFL1 (MFT)-like, each with distinct roles in plant growth and reproductive transitions [3]. FT-like proteins, including FT and its close homolog TWIN SISTER OF FT (TSF), serve as primary activators of flowering [9]. FT, often referred to as the florigen, acts as a mobile signal that travels from the leaves to the shoot apical meristem (SAM), where it interacts with the transcription factor FLOWERING LOCUS D (FD) to induce flowering [1,10]. TSF carries out a similar function; its overexpression accelerates flowering, whereas TSF-deficient mutants exhibit delayed flowering [11]. Conversely, TFL1-like proteins, including TFL1, BROTHER OF FT AND TFL1 (BFT), and CENTRORADIALIS (CEN), function as floral repressors [12]. TFL1 is primarily expressed in the inflorescence meristems, where it negatively regulates inflorescence development. Mutations in TFL1 result in accelerated flowering, emphasizing its role as a flowering inhibitor [1]. BFT, which exhibits diurnal expression patterns, delays flowering when overexpressed, although its knockdown has no significant effect, indicating functional redundancy within this subfamily [13]. In contrast, the MFT-like subfamily demonstrates attenuated FT-like activity and is primarily involved in seed germination and responses to environmental stress [14,15]. Interestingly, despite their high DNA sequence similarity, FT and TFL1 have opposing effects on flowering, reflecting the intricate regulatory mechanisms and evolutionary divergence within the PEBP family [16]. It is hypothesized that MFT-like genes may be ancestral to both FT-like and TFL1-like subfamilies, contributing to their functional diversification in plant developmental processes.
Elucidating the functions of the PEBP gene family is crucial for crop improvement, especially in relation to flowering time and plant architecture, two key factors that influence yield [17,18]. For instance, in rice, the florigen gene Hd3a exhibits approximately 70% amino acid sequence similarity with FT and 50% with TFL1 from Arabidopsis, and it enhances branching while promoting flowering [19]. In sesame, the TFL1-like gene SiPT1 significantly influences plant architecture; its overexpression results in increased branching while delaying flowering [20]. Similarly, in pepper (Capsicum spp.), two FT-like genes, CaFT5 and CaFT6, have been identified as key regulators of flowering time and plant structure [8]. Collectively, these findings highlight the diverse and vital roles of the PEBP gene family in regulating both plant growth and reproductive processes.
Sunflower (Helianthus annuus L.) is one of the most recognizable members of the Asteraceae family, which accounts for approximately 10% of all flowering plant species. It is the fourth most important oilseed crop globally, following palm, soybean, and rapeseed [21,22]. Furthermore, sunflower holds economic importance in the cut flower industry. Enhancing seed yield from terminal flower discs and improving terminal flower quality are the primary goals in both oilseed and ornamental sunflower breeding programs [23]. Plant architecture, specifically the distinction between uniculm and branched types, is a critical trait in sunflower cultivation. The presence of side buds and branches can negatively affect terminal flower development and increase production costs [23]. Consequently, developing new varieties with reduced or no lateral branching is vital for increasing seed yield. In ornamental sunflowers, branching patterns also play a critical role in determining flower quality and yield. Previous research has explored the role of recently duplicated FT paralogs in the sunflower genome during domestication, particularly focusing on the FT/TFL1 gene family [24,25]. These studies have revealed that modifications in gene expression, sequence variation, and gene interactions, notably a frameshift mutation in HaFT1, which is associated with a major QTL for flowering time, can delay flowering by interfering with HaFT4 [24,25]. These findings emphasize the importance of FT paralogs in driving adaptive changes, varied photoperiod responses, and evolutionary innovation through gene duplication. Despite these insights, the genome-wide characteristics and roles of PEBPs have not been systematically investigated in sunflower.
Thus, in this study, we conducted a comprehensive genome-wide identification and expression analysis of the HaPEBP gene family, employing bioinformatics approaches based on a self-constructed sunflower telomere-to-telomere (T2T) genome. The investigation encompassed an in-depth analysis of gene structure, selection pressure, and comparative evolutionary patterns within the HaPEBP family. The study also examined the physicochemical properties of HaPEBP proteins and predicted their 3D structural models. Furthermore, protein–protein interactions and potential regulatory networks were assessed using the STRING database. To further elucidate the functional roles of HaPEBP genes, their transcriptional profiles across various tissues were analyzed using RNA-seq data. These comprehensive analyses lay a robust foundation for understanding the biological functions and molecular regulatory mechanisms of the HaPEBP family, offering valuable insights into their functions in sunflower growth and development.

2. Results

2.1. Genome-Wide Identification and Phylogenetic Relationship of HaPEBP Members in Sunflower

Based on BLAST(version 2.8.1) and HMM (v3.3.2) searches, a total of 12 PEBP genes were identified in the T2T-assembled genome of sunflower, each containing the complete PEBP domain (Supplementary Table S1). These genes were sequentially named HaPEBP1 through HaPEBP12 according to their chromosomal positions in the T2T sunflower genome. The analysis and comparison of the CDS lengths, predicted protein sequences, MWs, isoelectric points (pI), subcellular localizations, and chromosomal distributions of the 12 genes were carried out (Supplementary Table S1). The CDS lengths ranged from 444 bp (HaPEBP11) to 648 bp (HaPEBP2), with an average length of 530 bp. Among them, HaPEBP2 encoded the longest PEBP protein with 215 amino acids, while HaPEBP11 encoded the shortest, comprising 147 amino acids. The MWs of these proteins ranged from 16.89 kDa to 24.50 kDa, with an average of 19.76 kDa. The pI values spanned from 4.91 (HaPEBP3) to 10 (HaPEBP8), with four HaPEBP proteins classified as acidic (pI < 7) and eight as basic (pI > 7). Further, all identified HaPEBP proteins were predicted to be hydrophilic, as evidenced by their negative Grand Average of Hydropathicity (GRAVY) values, with HaPEBP11 exhibiting the highest hydrophilicity.
To better understand the evolutionary relationships of the HaPEBP gene family, a phylogenetic tree was constructed using the 12 sunflower PEBP proteins alongside 17 homologs from rice, 8 from sesame, 13 from sorghum, and 6 from A. thaliana (Figure 1, Supplementary Table S2). The analysis classified the 68 PEBP proteins into three major subfamilies: 30 genes in the FT-like subgroup, 24 in the TFL1-like subgroup, and 14 in the MFT-like subgroup.

2.2. Conserved Motifs and Cis-Acting Elements of HaPEBP Genes

To gain further insights into the phylogenetic relationship of HaPEBP genes in sunflower, a phylogenetic tree was constructed using the Maximum Likelihood (ML) method via FastTree [26]. The resulting tree topology closely mirrored the broader phylogenetic tree presented in Figure 1, except for the TFL1-like subgroup, which formed two distinct clusters (Figure 2A). The HaPEBP family comprised 4 FT-like, 5 TFL1-like, and 3 MFT-like genes, consistent with previous gene classification results (Supplementary Table S2).
Subsequently, an analysis of conserved motifs on the 12 HaPEBP proteins harboring the PEBP domain was performed using the MEME program (Figure 2B). This analysis identified ten distinct motifs, labeled Motif 1 to Motif 10. Motifs 1 to 6 were highly conserved across the family, with Motif 1 appearing in all 12 HaPEBP genes. Within the FT-like subgroup, nearly all four members possessed six motifs: HaPEBP4, HaPEBP9, and HaPEBP10 contained Motifs 1–6, while HaPEBP8 lacked Motifs 2 and 5 but possessed Motif 8. The TFL1-like subgroup displayed greater motif diversity, with each gene containing between four and six motifs. The MFT-like subgroup showed the highest variability: HaPEBP1 included Motifs 1, 5, 6, and 10; HaPEBP2 featured Motifs 1, 5, 6, 8, and 10; and HaPEBP3 contained only Motif 1.
Given the crucial role of cis-acting elements in regulating gene expression, the 2000 bp upstream regions of the HaPEBPs were analyzed using the PlantCARE database to predict potential cis-acting elements (Figure 2C, Supplementary Table S3). The analysis detected 2109 occurrences of 77 distinct cis-acting element types across the promoter regions of 12 HaPEBPs. The number of cis-acting elements per gene ranged from 134 in HaPEBP8 to 301 in HaPEBP9, with an average of 176 elements per gene. These elements were classified into seven functional categories: hormone-responsive, growth- and development-related, stress-responsive, light-responsive, site-binding, promoter-related, and other functional elements. A majority of HaPEBP promoters featured CAAT or TATA boxes, which are typical cis-acting elements in promoter and enhancer regions (Figure 2C). Furthermore, 35 distinct cis-acting elements, including ABRE, G-Box, and Box4, were linked to responses involving light, hormone, promoter activity, and developmental processes. The widespread distribution of these elements across HaPEBP promoters indicates their potential roles in diverse biological processes.
To better understand the compositions and potential functions of the 12 HaPEBP genes, we analyzed their gene structures, conserved sequences, and exon/intron organization (Supplementary Figure S1, Supplementary Table S1). The analysis revealed that the number of exons per gene varied from 2 to 4. Interestingly, eight HaPEBP genes (67%) featured four exons and three introns, while the others had fewer, implying an intricate RNA splicing process. Within the FT-like subgroup, all four genes consistently possessed four exons and three introns. Similarly, most of the five TFL1-like genes shared this structure, except for HaPEBP7, which had only two exons and one intron. In contrast, members of the MFT-like subgroup exhibited fewer exons and introns: two genes contained three exons and two introns, while HaPEBP3 had just two exons and one intron. These observations suggest that, despite high conservation in the gene structures and protein motifs within the HaPEBP family, substantial divergence has occurred during evolutionary.

2.3. Chromosomal Localization and Gene Ontology Analysis of HaPEBP Genes

Utilizing the T2T genome of sunflower, a chromosomal mapping and comparative analysis of HaPEBP genes was conducted for the first time. The analysis showed that the 12 HaPEBP genes were unevenly distributed across the six chromosomes, with the remaining 11 chromosomes lacking any HaPEBP genes (Figure 3A). Notably, chromosome 17 contained three HaPEBP genes, while chromosomes 2, 9, 12, and 16 each carried two HaPEBP genes. Only one HaPEBP gene was found on chromosome 7. Six HaPEBP genes were transcribed in the same direction as the reference genome, whereas the other six were transcribed in the opposite direction.
Subsequently, a homolog gene analysis of HaPEBP in sunflower was carried out using MCScanX software (Figure 3B). This analysis identified 88 homologous gene pairs within the HaPEBP gene family (Supplementary Table S4). Of these, nine genes were attributed to tandem duplication events. HaPEBP4 and HaPEBP5, located on chromosome 9, were found to originate from dispersed duplication blocks, while HaPEBP3 on chromosome 7 arose from a dispersed duplication event (Supplementary Table S6). These findings indicate that tandem duplication events have played a crucial role in the evolutionary expansion of the HaPEBP gene family in sunflower.
Additionally, a conserved collinear analysis of the PEBP gene family across sunflower, Arabidopsis, and rice was carried out (Figure 3C). Specifically, 44 orthologous gene pairs were identified between sunflower (HaPEBPs) and Arabidopsis (AtPEBPs), with each AtPEBP having two or more corresponding orthologs in the sunflower genome. Notably, At_FT and At_TFL1 possessed over ten orthologous copies in sunflower (Supplementary Table S6). Furthermore, 108 orthologous gene pairs were found between sunflower (HaPEBPs) and rice (OsPEBPs), a number exceeding those found between sunflower and Arabidopsis. This difference is likely attributed to the higher number of OsPEBPs (12) compared to AtPEBPs (6). The identification of these orthologous genes, with potential similar functions, provided valuable insights into the characteristics of HaPEBPs.

2.4. Expression Characterization Analysis of HaPEBP Genes in Different Sunflower Tissues

To explore the expression profiles and potential functions of HaPEBP genes, the transcriptome data from five different tissues (root, stem, leaf, bud, and flower) of the sunflower cv. ‘Huoli’ were analyzed (Figure 4). Out of the 12 identified HaPEBP genes, HaPEBP1 was not expressed in any of the tissues examined. The other 11 genes exhibited the tissue-specific expression patterns (Figure 4). Notably, HaPEBP6 and HaPEBP11 demonstrated the highest expression levels in flower tissue. Additionally, five HaPEBP genes (HaPEBP4, HaPEBP5, HaPEBP7, HaPEBP8, and HaPEBP9) were predominantly expressed in the stem, while HaPEBP3 showed elevated expression in the bud tissue, implying a potential role in regulating shoot branching.
To confirm the accuracy of the transcriptome-based expression data, four HaPEBP genes (HaPEBP3, HaPEBP4, HaPEBP6, and HaPEBP9) were selected for qRT-PCR analysis. Expression levels were assessed in four different tissues across both uniculm-type germplasms (24DG080 and 24DG051) and branching-type germplasm (24FZ087) (Figure 5). The qRT-PCR results aligned with the transcriptome findings, showing that HaPEBP3 and HaPEBP9 were highly expressed in the leaves and flowers of the uniculm-type line 24DG051. In contrast, HaPEBP4 maintained low expression in the stem, leaves, and flowers of the branching-type line 24FZ087. Additionally, HaPEBP6 consistently exhibited low expression in the stem, leaves, and flowers across all three sunflower materials. These findings indicate that the HaPEBP gene family displays tissue-specific and genotype-dependent expression patterns, suggesting a potential regulatory role in shaping plant architecture among different sunflower varieties.

2.5. Subcellular Localization Analysis of HaPEBPs

Subcellular localization predictions using DeepLoc 2.0 indicated that the majority of HaPEBP proteins (10 out of 12) were located in the cytoplasm. However, two proteins (HaPEBP1 and HaPEBP3) were predicted to have dual location in both the cytoplasm and nucleus (Supplementary Table S7). Subsequent experimental validation through transient expression in tobacco confirmed that HaPEBP3 and HaPEBP9 were present in both the cytoplasm and nucleus (Figure 6).

2.6. Three-Dimensional Structure and Protein–Protein Interaction Analysis of HaPEBPs

To investigate the protein function of the HaPEBP gene family in sunflower, the three-dimensional (3D) structures of the 12 HaPEBP proteins were predicted using homology modeling based on their amino acid sequences and validated templates (Figure 7A, Supplementary Table S8). Structural analysis grouped the HaPEBP proteins into five distinct structural classes (Figure 7A), all of which shared common features such as multiple α-helices and extensive β-strands. Of these, eight proteins (67%) showed the structural similarity to the templates with PDB IDs 3AXY and 1QOU. HaPEBP1 aligned with the structure of 1WKP, HaPEBP3 exhibited similarity to 3N08, and HaPEBP6 and HaPEBP7 matched with 1WKO (Supplementary Table S8). To further explore the potential PPIs, an interaction network analysis was conducted using the STRING database (Figure 7B). The HaPEBP proteins showed extensive connections both within the family and with five other specific proteins (Figure 7B). Notably, several transcription factors (TFs), including FLOWERING LOCUS C (FLC) and TGACG-binding (TGA), exhibited differential expression across various tissues, indicating their involvement in key molecular mechanisms and biological processes related to flowering and development.

3. Discussion

The PEBP gene family plays a vital role in regulating key developmental and physiological processes in plants, including flowering time, plant architecture, floral organogenesis, seed germination, and responses to abiotic stress [2,17]. In sunflower, FT genes have been recognized as primary regulators of flowering time [27]. However, a comprehensive analysis of the entire HaPEBP gene family and its regulatory characteristics in sunflower has not yet been conducted.
In this study, 12 HaPEBP genes from the T2T sunflower genome were identified and characterized through combined HMM and BLAST search methods, with further validation through the PfamScan tool. All 12 HaPEBP genes were found to possess complete PBP domains and demonstrated evolutionary features. Expression profile analysis suggested that part of the HaPEBP genes might play various roles in regulating flowering time and plant architecture, offering valuable insights into the underlying molecular mechanisms governing these traits in sunflower.

3.1. Evolution Characteristics of HaPEBP Genes

In sunflower, the 12 identified HaPEBP genes display unevenly distribution across six chromosomes. Of these, nine HaPEBP genes exhibit evolutionary patterns primarily driven by tandem duplicated events, a common evolutionary mechanism that increases gene copy number and enables both functional diversification and sub-functionalization. Although whole-genome duplication (WGD) is prevalent in numerous plant species, our findings indicate that tandem duplications represent the primary force behind the expansion of the PEBP gene family in sunflower. This has led to higher numbers of HaPEBP genes compared to most eudicots, where this family typically contains fewer than ten genes [10].
Based on multi-alignments and phylogenetic analysis, the HaPEBP genes were categorized into three subgroups: TFL1-like, FT-like, and MFT-like (Figure 2A). Comparative phylogenetic analysis with other plant species, such as rice (17 OsPEBPs), sorghum (13 SbPEBPs), sesame (8 SiPEBPs), and A. thaliana (6 AtPEBPs), validated this classification (Figure 1), aligning with patterns observed in other dicot oilseed crops like sesame and peanut [20,28]. Notably, the classification of four sorghum PEBP (SbPEBP) genes was revised, with Sb_TFL3 and Sb_TFL2 being reclassified as FT-like, and Sb_FT7 and Sb_FT being reassigned to the TFL-like group (Figure 1, Supplementary Table S2). Additionally, 44 orthologous gene pairs were identified between HaPEBPs and AtPEBPs, and 108 pairs between HaPEBPs and OsPEBPs (Figure 3C).
In Arabidopsis, the FT gene acts as a key regulator of flowering time by encoding a protein (florigen) that, when transported from the leaves to the shoot apex, interacts with the basic leucine zipper (bZIP) transcription factor FD to activate floral meristem identity genes such as LEAFY (LFY) and APETALA1 (AP1), thus promoting flowering [29]. AtFT also interacts with the transcription factor BRANCHED1 (BRC1) to influence shoot branching [30]. Similarly, ectopic overexpression of pepper CaFT5 and CaFT6 in tobacco accelerates flowering, increases branching, and reduces leaf size, demonstrating their conserved roles in flowering promotion and plant form. CaFT5 is mainly expressed in shoot apical meristems and flowers, whereas CaFT6 is enriched in roots and fruits, implying redundant functions in branching and fruit development [8]. Conversely, the TFL1 gene suppresses flowering and preserves the inflorescence meristem identity by also interacting with FD. Loss of TFL1 function results in early flowering, whereas its overexpression delays flowering and inhibits terminal flower formation [1,31]. In rice, TFL1-related genes such as RCN1, Ghd7, and DTH8 also inhibit flowering. RCN1 delays flowering by competing with Hd3a for binding 14-3-3 proteins and OsFD1, while Ghd7 and DTH8 repress the expression of key flowering genes such as Ehd1, Hd3a, and RFT1 under long-day conditions, thereby delaying flowering [19,32]. Moreover, CRISPR/Cas9-mediated knockouts of FT-like and TFL1-like homologs in petunia yielded more compact, highly branched plants that flowered early, underscoring the parallel functions of PEBP subfamily members across angiosperms [33]. These observations suggest that the FT-like and TFL-like subfamily members of HaPEBP genes may have conserved roles similar to their orthologous counterparts in Arabidopsis, rice, and petunia, particularly in regulating flowering and shaping plant form.

3.2. Structure Variation of HaPEBP Genes

HaPEBP genes within the same subfamilies exhibited similar gene structures, while notable structural variations were observed among genes from different subfamilies (Figure 2B,C). Multiple sequence alignments indicated that all members of the sunflower PEBP gene possess the conserved motifs ‘D-P-d-x-P’ and ‘G-x-H-R’ (Supplementary Figure S2). Interestingly, the MFT subfamily exhibited substantial polymorphism across plants, particularly among angiosperms, where the MFT genes have evolved into distinct subgroups, reflecting their functional diversification and evolutionary adaptation [15].
The analysis of exon–intron structure revealed that 8 out of the 12 HaPEBP genes typically consist of four exons and three introns, an arrangement consistent with other PEBP genes [3,34]. However, exceptions were observed in genes like HaPEBP3 and HaPEBP7, each containing only two exons and a single intron (Supplementary Figure S1). Corresponding alterations were also noted in conserved protein motifs; for instance, HaPEBP3 retained only one motif, a pattern also documented in pepper [8]. The consistent motif distribution within each HaPEBP subfamily (Figure 2B) highlights their evolutionary conservation, while the observed variations point to a balance between conservation and divergence in gene function.
Cis-acting regulatory elements, typically located within 2000 base pairs upstream of a gene, play a crucial role in modulating gene expression. These elements are instrumental in plant responses to various abiotic stresses and are integral to growth and developmental processes across diverse crop species [35]. In the putative promoters of PEBP genes, cis-acting elements have been shown to mediate responses to abiotic and biotic stresses, regulate hormone-responsive transcription, and coordinate key processes in plant growth and development [7]. Typical light response elements, such as GT1-motif, Box 4, G-box, and TCT-motif, which could be analyzed similarly to the FT expression assessment in pears and Dendrobium species [36,37]. These elements mediate the response to light, influencing flowering time and photoperiod sensitivity. Additionally, the various HaPEBP members exhibit characteristics associated with different plant hormones, including ABRE for abscisic acid (ABA), AuxRR-core and TGA-element for auxin, CGTCA-motif for methyl jasmonate (MeJA), GARE-motif and TATC-box for gibberellin, and TCA-element for salicylic acid (SA). These findings suggest that HaPEBP genes likely contribute to the regulation of plant hormone responses.
Collinearity analysis within sunflower revealed 88 intra-species gene pairs, with the majority of HaPEBP genes demonstrating clear evidence of tandem duplication (Figure 3B, Supplementary Table S5). The analysis of Ka/Ks ratios for all HaPEBP genes consistently produced values less than 1 (Supplementary Table S9), suggesting that purifying selection has preserved both the structure and function of this gene family. Although tandem duplication, WGD, and segmental duplication are all established drivers of plant genome evolution [38,39], the findings indicate that tandem duplication is the primary mechanism driving the expansion and diversification of the HaPEBP gene family in sunflower.

3.3. Biological Functions of HaPEBP Genes

Several studies have shown that the expression patterns of PEBP genes are closely associated with their biological functions. For instance, in A. thaliana, the FT gene (AtFT) is expressed in both reproductive and vegetative organs, where it promotes flowering in conjunction with flowering-related LFY gene [40]. Similarly, this study found that HaPEBP genes exhibited tissue-specific expression, indicating their involvement in various aspects of growth and development. Notably, HaPEBP9 and HaPEBP10, both members of the FT-like subfamily, were predominantly expressed in leaves (Figure 4). Their promoter regions contained an abundance of light-responsive elements, including Sp1, G-box, and Box-4, suggesting that these genes may be light-sensitive and play significant roles in controlling flowering time [41,42]. Another FT-like gene, HaPEBP8, showed predominant expression in stems, indicating its potentially important role in developmental regulation. These FT-like genes were expressed across a range of tissues, and their response elements reflected this diversity (Figure 3C). This widespread tissue expression suggests that the HaPEBP genes have multifunctional roles in sunflower growth and development. Similar to AtFT, which encodes florigen proteins essential for reproductive transition and plant architecture, the homologous HaPEBP genes likely participate in a comparable regulatory process. Phylogenetic analysis revealed that the three FT-like genes shared a high degree of amino acid similarity with AtFT [43,44], with only minimal differences (Figure 1). Furthermore, the three HaPEBP proteins were localized in the cytoplasm, a characteristic also observed in AtFT and the cotton homologous proteins GhFT [45].
Plant architecture is a critical trait that significantly influences both the development of terminal flowers and the costs of sunflower production [23]. The TFL1-like gene, in particular, plays a crucial role in regulating the transition from vegetative to reproductive growth, thereby determining whether plants exhibit indeterminate or determinate growth habits [46]. For example, in Arabidopsis, TFL1 sustains indeterminate growth by suppressing genes responsible for floral meristem identity, and mutations in the TFL1-like gene lead to early flowering and a switch to determinate growth [1]. A similar function is noted in sesame, where the TFL1-like subfamily gene SiCEN2 influences plant form and inflorescence development. Variations in SiCNE2, such as LTR insertions, significantly impact branching and the timing of flowering [20]. In other crops like pigeon peas and rapeseed, mutations in TFL1-like homologs are associated with variations in branching, plant height, and flowering time, emphasizing the roles of TFL1-like genes in shaping growth habits [47,48]. Moreover, gene duplications and the subsequent functional divergence within the TFL1-like gene family help drive the evolution of plant architectures by modulating flowering and growth in response to environmental cues. In the present study, phylogenetic analysis revealed that five HaPEBP genes (HaPEBP5, HaPEBP6, HaPEBP7, HaPEBP11, and HaPEBP12) belonged to the TFL1-like subfamily and clustered closely with established regulators such as SiCEN2 and ATCEN (Figure 1), suggesting that they may play similar roles in shaping plant architecture in sunflower. Transcriptome analysis further substantiated this observation, as these genes were highly expressed in key tissues, namely the stem, flower, and bud. Specifically, HaPEBP3 demonstrated elevated expression in the stem and bud, aligning with previous studies highlighting its regulatory role in plant architecture [49]. Collectively, these findings offer valuable insights into the genetic regulation of plant architecture and denote candidate target genes for sunflower breeding efforts focused on optimizing plant architecture.
This article examined the 3D structure of HaPEBP proteins to gain deeper insights into the structural features and potential functions of the HaPEBP proteins in sunflower. Based on data from the RCSC PDB database, five comparative homology models for 12 HaPEBP proteins were available. The sequence identity ranged from 35% to 81% (Supplementary Table S8). All five models exhibited characteristic PEBP structural elements, such as multiple α-helices and a prominent β-sheet (Figure 7A). Notably, 67% of HaPEBP proteins showed structural similarity to templates with PDB IDs 3AXY and 1QOU, which correspond to PEBP protein from rice (OsFD3) and Antirrhinum, respectively [50,51]. Specific alignments were also observed: HaPEBP1 aligned with portions of the structure represented by PDB ID 1WKP, HaPEBP3 with PDB ID 3N08, and both HaPEBP6 and HaPEBP7 with PDB ID 1WKO [52,53]. These structural similarities indicate a conserved fold among HaPEBPs, likely critical for their function. This conserved structure suggests a preserved molecular mechanism underlying their biological functions and highlights their potential involvement in key regulatory pathways across plant species.
The PPI network analysis of HaPEBP proteins in sunflower, conducted using the STRING database, demonstrates the high conservation and central regulatory roles of PEBP proteins in plant development (Figure 7B). A confidence score threshold of 0.4 was used to ensure reliable interactions among the proteins. Within this extensively connected network, YggU, a plant cysteine protease, emerges as a notable interactor. Cysteine proteases play multifaceted roles in plants, including response to pathogens, growth, development, senescence, programmed cell death, and the accumulation and mobilization of storage proteins, as well as responses to biotic and abiotic stresses [54]. The elevated expression of YggU across various sunflower tissues indicates its substantial role in these physiological processes. Another significant interactor is the FLC protein, a MIKC-type MADS transcription factor, that interacts with FT and other PEBP family members. FLC is essential for regulating flowering time and plant development by suppressing flowering integrator genes, thus controlling the transition from vegetative to reproductive stages [55]. Additionally, TGA transcription factors, part of the bZIP family, play vital roles in plant immune responses and developmental processes. Their interactions with PEBP family proteins facilitate the integration of signals from diverse pathways, enabling precise regulation of plant growth, development, and flowering. For instance, TGA7 regulates flowering time by modulating flowering repressor genes, including FLC [56]. Furthermore, TGA transcription factors influence plant flowering time and organ development through interactions with PEBP family proteins [57]. The different expression levels of these transcription factors and their interactions with HaPEBP proteins reflect a complex regulatory network governing flowering and developmental processes in sunflower. Future investigation into the key HaPEBP genes in this interaction network may offer deeper insights into their specific roles and regulatory pathways. This study will enhance our understanding of PEBP gene functions in sunflower and other crops, offering potential strategies for improving plant architecture and yield through targeted breeding.

4. Methods and Materials

4.1. Plant Materials

This study utilized three sunflower germplasm accessions for gene expression analysis: 24DG080 and 24DG051 (uniculm-type), and 24FZ087 (branching-type). All materials were cultured at the Yuanyang experimental station (113°97′ E and 35°05′ N), where their phenotypes were continuously monitored and samples collected during the 2023–2024 growing season. The sunflower materials were supplied by the Economic Crop Research Institute of Henan Academy of Agricultural Sciences (ECRI-HAAS), Zhengzhou, Henan, China. Seeds were sown in the field on 7 June 2024, following standard agronomic practices. Tissue samples, including root, stem, leaf, and flower, were collected at the onset of flowering (19 July 2024), immediately frozen in liquid nitrogen, and stored at −80 °C until further analysis.

4.2. Identification of the PEBP Gene Family in Sunflower

This study employed the first assembled T2T genome of sunflower (Chinese variety, Sandaomei), which is publicly available in the National Genomics Data Center (NGDC) database (project no. PRJCA028800). PEBP protein sequences from Arabidopsis thaliana, retrieved from The Arabidopsis Information Resource (TAIR) database, served as queries in a BLASTP search against the sunflower protein sequences, using an E-value threshold of <1 × 10−5 and default parameters. Additionally, the seed file for the PEBP domain (PF01161) was acquired from the Protein Families Database (Pfam, v35.0) [58]. The HMMER software (v3.3.2) was employed to identify PEBP domains within the sunflower protein sequences [59]. Candidate genes were further validated for the presence of the PEBP domain using PfamScan (v1.6) [60], and only those containing a complete conserved PEBP domain were retained for further analysis.

4.3. Multiple Sequence Alignment and Phylogenetic Analysis of HaPEBP

Before exploring the diversity of HaPEBPs, full-length amino acid sequences of PBEPs from Oryza sativa, A. thaliana, Sesamum indicum, and Sorghum bicolor were retrieved from previous studies [6,7,20,34]. These sequences were aligned using MAFFT (v7.505) [61], and a phylogenetic tree was constructed using the Maximum Likelihood (ML) method in FastTree v2.1.1. The analysis employed the Jones–Taylor–Thornton (JTT) amino acid substitution model, the default setting in FastTree for protein sequence alignments, selected for its effectiveness in analyzing moderately divergent protein sequences. Tree reliability was assessed through 1000 bootstrap replications [26]. The phylogenetic tree for HaPEBPs was visualized using FigTree (v1.4.4, available online: http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 20 June 2021).

4.4. Motifs and Gene Structure Analysis

The conserved motifs within each HaPEBP member were analyzed using the Multiple Expectation Maximization for Motif Elicitation (MEME) Suite (version 5.5.7), with parameters configured to identify a maximum of 10 motifs [62]. The gene structures of HaPEBPs were analyzed via the online Gene Structure Display Server (GSDS 2.0) [63].

4.5. Identification and Analysis of Cis-Acting Elements in Gene Promoters

The 2000 bp upstream sequence of the HaPEBP genes was extracted as the putative promoter regions and analyzed for cis-acting elements using the default settings in the PlantCARE database [64]. The results were then visualized using TBtools (version 2.210) [65].

4.6. Characterization, Chromosomal Localization, and Synteny Analysis of HaPEBP Genes

The genomic positions of HaPEBP genes were mapped using the MapGene2Chrom (v2.1) platform [66]. The sequence length, molecular weight (MW), and isoelectric point (pI) were calculated using tools available on the ExPasy website [67]. Pairwise genome comparisons among A. thaliana, O. sativa, and sunflower were performed using the JCVI software v1.4.24 [68]. Synteny regions and chromosome distributions were analyzed using MCScanX v1.0.0 with default parameters, and the results were visualized using Circos v0.52 [69].

4.7. Analysis of Selective Pressures on HaPEBP Genes

Homology alignment of HaPEBP protein sequences was first carried out and subsequently translated the resulting alignment into a codon-level alignment. Pairwise nonsynonymous (Ka) and synonymous (Ks) substitution rates were then calculated using KaKs_Calculator (V3.0) [70], which employs model selection and averaging methods to estimate these rates. The Ka/Ks ratio was analyzed to evaluate the selective pressures on gene duplication events among homologous HaPEBP genes in sunflower.

4.8. Subcellular Localization

The subcellular localization of HaPEBP proteins was predicted using the DeepLoc 2.0 online tool [71]. The coding sequence (CDS) of the target gene (without the stop codon) was amplified from sunflower materials and ligated to the N-terminus of GFP within the pCambia1300 vector to construct the 35S:Gene-GFP recombinant expression vector. The constructed plasmids were transformed into the Agrobacterium tumefaciens GV3101 strain and transiently expressed in Nicotiana benthamiana leaves via the infiltration method. Following 8 h of incubation in the dark, the plants were moved to normal light conditions for an additional 48 h. The GFP fusion protein fluorescence signal was detected and imaged using a Nikon laser scanning confocal microscope (NiKon-AX, NIS-Elements 5.4, Tokyo, Japan). Primers used for amplification are listed in the Supplementary Table S10.

4.9. Expression Profile Assay of HaPEBP Gene and RNA-Seq Data Analysis

To investigate the expression patterns of HaPEBP genes across different sunflower tissues, the Fragments Per Kilobase of exon model per Million mapped reads (FPKM) matrix data for the sunflower cv. ‘Huoli’ were obtained from the NCBI database (accession number GSE221055) [49]. The log2 fold change (log2FC) for each treatment was calculated and visualized as heatmaps using the R package ‘pheatmap’ (version 1.0.12).
Total RNA was isolated from various sunflower tissues (root, stem, leaf, bud, and seed) across three germplasm accessions, using the Polysaccharide Polyphenol Total RNA Kit (Tiangen, Beijing, China). The quality of the RNA samples was assessed using a spectrophotometer (Nanodrop, Thermo Scientific, Waltham, MA, USA) and by performing agarose gel electrophoresis. cDNA synthesis was carried out using DNase I (Thermo Scientific, #EN0521) and the RevertAid™ First Strand cDNA Synthesis Kit (Thermo Scientific, #K1622). The β-tubulin gene served as the internal reference gene for quantitative real-time PCR (qRT-PCR) analysis. The relative expression levels of HaPEBP genes were calculated using the 2−ΔΔCt method [72]. qRT-PCR was conducted using the Roche LightCycler® 480 II Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). Primer pairs specific to HaPEBP alleles (Supplementary Table S10) were designed using Primer Premier 5.0 (available online: http://www.premierbiosoft.com/primerdesign/index.html (accessed on 8 January 2021). All reactions were performed in triplicate, and the gene expression levels were normalized against the β-tubulin gene.

4.10. Analysis of the Three-Dimensional Structure and Protein–Protein Interactions of HaPEBP Proteins

To investigate the three-dimensional (3D) structure of HaPEBP proteins, comparative modeling was performed using the RCSB Protein Data Bank (PDB) [73]. The resulting 3D structures were visualized and examined using PyMOL (v2.5.4) [74]. PPIs were predicted using the STRING database v10 [75], employing an interaction score threshold of 0.4 to ensure high confidence in the predicted interactions. The resulting PPI networks were then visualized and analyzed using Cytoscape (v3.9.1) [76].

5. Conclusions

In this study, we identified 12 HaPEBP genes within the sunflower T2T genome and performed a comprehensive analysis of their chromosomal distribution, phylogenetic relationships, gene structures, and expression profiles. These genes were categorized into three distinct subfamilies: TFL1-like, FT-like, and MFT-like, each characterized by conserved sequence motifs and largely influenced by tandem duplication events. The analysis of the promoter regions of 12 HaPEBP genes revealed 77 distinct types of cis-acting elements, with 35 elements potentially associated with light responsiveness and plant growth and development. Expression profiling based on RNA-seq and RT-PCR indicated that HaPEBP genes exhibit tissue-specific expression patterns. PPI network analysis further confirmed their central roles in regulating flowering and plant architecture. Collectively, these findings shed light on the structure, evolution, and functional diversification of the HaPEBP gene family and provide valuable genetic resources for the development of sunflower cultivars with adaptive flowering time and optimized plant architecture.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms26104602/s1.

Author Contributions

Conceptualization, H.C., X.Y. and L.L.; Data curation, Y.S., G.L., Q.T. and H.C.; Formal analysis, G.L., Q.T. and S.L.; Funding acquisition, Y.S. and L.L.; Investigation, Y.S., Y.W., J.B. and J.G.; Methodology, Y.S. and Y.W.; Project administration, Y.S., H.C. and L.L.; Resources, S.L. and X.Y.; Software, Y.S., Y.W., J.B., J.G. and H.C.; Supervision, H.C. and L.L.; Validation, Y.S.; Visualization, Y.S., G.L., Q.T. and H.C.; Writing—original draft, Y.S.; Writing—review and editing, H.C., X.Y. and L.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the College Student Innovation and Entrepreneurship Training Program of Henan University, grant no. XJ2024225.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are in the article/Supplementary Materials, and further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree of PEBP genes among five plants. The 68 PEBP genes, including 12 HaPEBP genes from sunflower (H. annuus), 17 OsPEBPs from rice (O. sativa), 6 AtPEBPs from A. thaliana, 8 SiPEBPs from sesame (S. indicum), and 13 SbPEBPs from sorghum (S. bicolor), are classified into three subgroups, i.e., FT-like (in green), TFL1-like (in blue), and MFT-like (in white).
Figure 1. Phylogenetic tree of PEBP genes among five plants. The 68 PEBP genes, including 12 HaPEBP genes from sunflower (H. annuus), 17 OsPEBPs from rice (O. sativa), 6 AtPEBPs from A. thaliana, 8 SiPEBPs from sesame (S. indicum), and 13 SbPEBPs from sorghum (S. bicolor), are classified into three subgroups, i.e., FT-like (in green), TFL1-like (in blue), and MFT-like (in white).
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Figure 2. Phylogenetic and conserved motif analysis of HaPEBP genes. (A) Phylogenetic tree of the 12 HaPEBP proteins constructed using the Maximum Likelihood (ML) method. The 12 HaPEBPs are divided into four distinct groups, i.e., FT (in blue), TFL (in deep blue), MFT (in red), and BFT (in vermilion). (B) Distribution of conserved motifs within each HaPEBP protein. Ten distinct motifs, named Motifs 1 to 10, are shown in colorful blocks. The black scale bar at the bottom represents the distance between motifs. (C) Distribution of cis-acting elements related to light response and plant growth and development in the putative promoters of HaPEBP genes. A total of 31 cis-acting elements are identified in the promoters of the 12 HaPEBP genes. The 12 most common elements in HaPEBP genes are shown in different segments in the figure. The black scale indicates the sequence position of HaPEBP promoters.
Figure 2. Phylogenetic and conserved motif analysis of HaPEBP genes. (A) Phylogenetic tree of the 12 HaPEBP proteins constructed using the Maximum Likelihood (ML) method. The 12 HaPEBPs are divided into four distinct groups, i.e., FT (in blue), TFL (in deep blue), MFT (in red), and BFT (in vermilion). (B) Distribution of conserved motifs within each HaPEBP protein. Ten distinct motifs, named Motifs 1 to 10, are shown in colorful blocks. The black scale bar at the bottom represents the distance between motifs. (C) Distribution of cis-acting elements related to light response and plant growth and development in the putative promoters of HaPEBP genes. A total of 31 cis-acting elements are identified in the promoters of the 12 HaPEBP genes. The 12 most common elements in HaPEBP genes are shown in different segments in the figure. The black scale indicates the sequence position of HaPEBP promoters.
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Figure 3. Syntenic distribution of HaPEBP genes in the sunflower genome. (A) Distribution and chromosomal localization of HaPEBP genes. Orange column represents sunflower chromosomes. (B) Synteny analysis and distribution pattern of HaPEBP genes. Red line indicates the syntenic gene pair within the HaPEBP gene family. (C) Collinearity analysis of PEBP genes among the genomes of Arabidopsis, sunflower, and rice. The chromosomes of Arabidopsis, sunflower, and rice are shown in black, orange, and green segments, respectively. Gray lines represent the overall gene collinearity between two crops. Red lines highlight the collinearity specific to the PEBP gene family.
Figure 3. Syntenic distribution of HaPEBP genes in the sunflower genome. (A) Distribution and chromosomal localization of HaPEBP genes. Orange column represents sunflower chromosomes. (B) Synteny analysis and distribution pattern of HaPEBP genes. Red line indicates the syntenic gene pair within the HaPEBP gene family. (C) Collinearity analysis of PEBP genes among the genomes of Arabidopsis, sunflower, and rice. The chromosomes of Arabidopsis, sunflower, and rice are shown in black, orange, and green segments, respectively. Gray lines represent the overall gene collinearity between two crops. Red lines highlight the collinearity specific to the PEBP gene family.
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Figure 4. Expression profiles of the 12 HaPEBP genes in different tissues. Sunflower tissues, including root, stem, leaf, bud, and flower, are applied for gene expression analysis. The color scale represents the log2-transformed FPKM value. The high and the low expression levels of each gene are individually shown in dark red and bright green, respectively.
Figure 4. Expression profiles of the 12 HaPEBP genes in different tissues. Sunflower tissues, including root, stem, leaf, bud, and flower, are applied for gene expression analysis. The color scale represents the log2-transformed FPKM value. The high and the low expression levels of each gene are individually shown in dark red and bright green, respectively.
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Figure 5. Comparison of HaPEBP gene expression and phenotypic characteristics across three sunflower varieties. (A) Phenotypic images of three sunflower germplasm accessions: 24DG051 (uniculm-type, left), 24DG080 (uniculm-type, middle), and 24FZ087 (branching-type, right). The top panel shows plant images at the pre-flowering stage, and the bottom panel displays flower images at the full-bloom stage for each variety. (B) Relative expression levels of four HaPEBP genes in the three sunflower varieties. The bar represents the mean ± standard error (SE) from three independent biological replicates. The germplasm accessions are indicated by color: 24FZ087 (branching-type) in blue, 24DG051 (uniculm-type) in orange, and 24DG080 (uniculm-type) in green.
Figure 5. Comparison of HaPEBP gene expression and phenotypic characteristics across three sunflower varieties. (A) Phenotypic images of three sunflower germplasm accessions: 24DG051 (uniculm-type, left), 24DG080 (uniculm-type, middle), and 24FZ087 (branching-type, right). The top panel shows plant images at the pre-flowering stage, and the bottom panel displays flower images at the full-bloom stage for each variety. (B) Relative expression levels of four HaPEBP genes in the three sunflower varieties. The bar represents the mean ± standard error (SE) from three independent biological replicates. The germplasm accessions are indicated by color: 24FZ087 (branching-type) in blue, 24DG051 (uniculm-type) in orange, and 24DG080 (uniculm-type) in green.
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Figure 6. Subcellular localization of the HaPEBP-GFP fusion protein in N. benthamiana leaves. GFP alone is used as a control. Nuclei are counterstained with DAPI.
Figure 6. Subcellular localization of the HaPEBP-GFP fusion protein in N. benthamiana leaves. GFP alone is used as a control. Nuclei are counterstained with DAPI.
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Figure 7. Three-dimensional structure and PPI networks of HaPEBP proteins. (A) Five structure models of HaPEBPs. The models are determined using RCSC PDB. (B) PPI analysis of HaPEBP proteins. The network is conducted using the STRING database.
Figure 7. Three-dimensional structure and PPI networks of HaPEBP proteins. (A) Five structure models of HaPEBPs. The models are determined using RCSC PDB. (B) PPI analysis of HaPEBP proteins. The network is conducted using the STRING database.
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Sun, Y.; Wang, Y.; Bai, J.; Guo, J.; Li, G.; Tian, Q.; Lv, S.; Cao, H.; Yang, X.; Liu, L. Genome-Wide Identification and Expression Profiling of Phosphatidylethanolamine-Binding Protein (PEBP) Genes in Helianthus annuus L. Int. J. Mol. Sci. 2025, 26, 4602. https://doi.org/10.3390/ijms26104602

AMA Style

Sun Y, Wang Y, Bai J, Guo J, Li G, Tian Q, Lv S, Cao H, Yang X, Liu L. Genome-Wide Identification and Expression Profiling of Phosphatidylethanolamine-Binding Protein (PEBP) Genes in Helianthus annuus L. International Journal of Molecular Sciences. 2025; 26(10):4602. https://doi.org/10.3390/ijms26104602

Chicago/Turabian Style

Sun, Yiyi, Yanwen Wang, Jingyan Bai, Jiatong Guo, Guiting Li, Qiuzhen Tian, Shuping Lv, Hengchun Cao, Xiaojie Yang, and Lingyun Liu. 2025. "Genome-Wide Identification and Expression Profiling of Phosphatidylethanolamine-Binding Protein (PEBP) Genes in Helianthus annuus L." International Journal of Molecular Sciences 26, no. 10: 4602. https://doi.org/10.3390/ijms26104602

APA Style

Sun, Y., Wang, Y., Bai, J., Guo, J., Li, G., Tian, Q., Lv, S., Cao, H., Yang, X., & Liu, L. (2025). Genome-Wide Identification and Expression Profiling of Phosphatidylethanolamine-Binding Protein (PEBP) Genes in Helianthus annuus L. International Journal of Molecular Sciences, 26(10), 4602. https://doi.org/10.3390/ijms26104602

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