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. 2025 Aug 7;14(8):1012. doi: 10.3390/biology14081012

Characterization and Functional Analysis of the FBN Gene Family in Cotton: Insights into Fiber Development

Sunhui Yan 1,†, Liyong Hou 2,†, Liping Zhu 3, Zhen Feng 1, Guanghui Xiao 3,*, Libei Li 1,*
Editors: Robert Henry, Miki Daisuke
PMCID: PMC12383549  PMID: 40906207

Simple Summary

The FBN gene family in cotton was characterized in this study: 28 members were identified in upland cotton (Gossypium hirsutum), with comparable numbers in other cotton species. Allotetraploid species harbor approximately twice as many FBN genes as diploids, indicating lineage-specific expansion during polyploidization. Phylogenetic and structural analyses showed these genes cluster into 11 groups, share conserved motifs and the PAP-fibrillin domain, and contain hormone-responsive cis-elements. Expression profiling revealed that specific GhFBN genes, such as GhFBN2-2, are highly expressed during key fiber development stages. Hormone treatments demonstrated that GhFBN1A-2 is induced by GA3, which promotes fiber growth, but repressed by MeJA, while GhFBN11-2 is downregulated by GA3 and IAA. These findings highlight FBNs’ role in hormone-mediated cotton fiber development, providing targets for improving fiber quality in breeding programs.

Keywords: cotton, FBN gene family, fiber development, hormone response, gene expression

Abstract

Fibrillins (FBNs) are indispensable for plant growth and development, orchestrating multiple physiological processes. However, the precise functional role of FBNs in cotton fiber development remains uncharacterized. This study reports a genome-wide characterization of the FBN gene family in cotton. A total of 28 GhFBN genes were identified in upland cotton, with systematic analyses of their phylogenetic relationships, protein motifs, gene structures, and hormone-responsive cis-regulatory elements. Expression profiling of GhFBN1A during fiber development revealed stage-specific activity across the developmental continuum. Transcriptomic analyses following hormone treatments demonstrated upregulation of GhFBN family members, implicating their involvement in hormone-mediated regulatory networks governing fiber cell development. Collectively, this work presents a detailed molecular characterization of cotton GhFBNs and establishes a theoretical foundation for exploring their potential applications in cotton breeding programs aimed at improving fiber quality.

1. Introduction

Fibrillin, a plastid lipid-associated protein (PAP) first identified in red rose (Rosa rugosa) and bell pepper (Capsicum annuum), is conserved across photosynthetic organisms spanning from cyanobacteria to higher plants [1,2,3,4,5,6,7]. Since its discovery, extensive studies have shown that fibrillin is remarkably conserved across a wide spectrum of photosynthetic organisms, ranging from simple cyanobacteria to complex higher plants. This conservation implies that fibrillin plays fundamental roles in photosynthetic processes that are essential for the survival and growth of these organisms. For example, in cyanobacteria, fibrillin proteins might be involved in maintaining the stability of the photosynthetic apparatus, which is crucial for their autotrophic lifestyle [8]. Fibrillins, initially characterized by their subcellular localization and functional specializations in distinct organelles, have been variously denominated as plastid lipid-associated protein (PAP), plastoglobule protein (PGL), chloroplast drought-induced stress protein of 34 kDa (CDSP34), and chromoplast-specific carotenoid-associated protein (ChrC) [2,3,9,10].

Research on the subcellular localization of FBN proteins is crucial for understanding their functions. Multiple studies have shown that FBN proteins are predominantly located in chloroplasts, distributed across the stroma, thylakoids, and plastoglobules (PGs) [6,11,12]. FBN proteins are also present in plastids other than chloroplasts, with relevant studies having elucidated their distribution in plastid-containing organisms and the significance of their presence in non-chloroplast plastids [4,13]. Overall, FBN proteins’ subcellular localization provides a spatial basis for their involvement in photosynthesis, lipid metabolism, plant development, and stress responses.

Several investigations have shown that FBNs centrally regulate key physiological processes in diverse plant species, including photosynthetic mechanisms, stress response, and plant growth and development [14,15,16,17]. Among the 14 FBN isoforms in Arabidopsis, FBN1a, FBN1b, and FBN2 regulate jasmonic acid biosynthesis under light or low-temperature stress [12,18]. In bell pepper, FBN1 protein levels correlate positively with fruit ripening, while in potato, high expression of C40.4 (FBN1) enhances tuber carotenoid content [19]. The OsFBN gene in rice is induced by extreme temperature stress [10,20]. In diverse plant species, including cucumber (Cucumis sativus), tomato (Solanum lycopersicum), sweet orange (Citrus sinensis) and mustard (Brassica juncea), the expression of FBN proteins has been reported [21,22,23,24].

Cotton, as the world’s premier natural fiber crop, serves as an ideal model system for studying cell elongation [25,26]. Gossypium hirsutum, domesticated approximately 8000 years ago, has become the predominant cultivated cotton species due to its production of relatively long fiber cells that are well-adapted to modern textile manufacturing processes [25]. The fiber quality of cotton directly affects the economic efficiency of the textile industry. Cotton fiber cell development is typically divided into five overlapping stages: cell initiation, elongation, transitional wall thickening, secondary cell wall thickening, and maturation [27]. The elongation stage is a critical period that determines fiber length, an important trait that directly influences the economic value of cotton [28]. Despite the established role of FBNs in plant development across multiple species, their function in cotton fiber development remains uncharacterized. Here, we identified the GhFBN gene family from four cotton species’ genomic data via bioinformatics, characterizing their structures, chromosomal locations, phylogeny, collinearity, and expression profiles to reveal functional diversity. This study deepens the understanding of cotton GhFBN genes, highlights their potential in enhancing fiber quality, and provides valuable candidate genes and theoretical support for molecular breeding to improve cotton fiber traits in agricultural practice.

2. Materials and Methods

2.1. Identification of Cotton GhFBN Genes

Amino acid sequences of the Arabidopsis FBN gene family were obtained from the Arabidopsis Information Resource (https://www.arabidopsis.org, (accessed on 5 April 2025)). Subsequently, these sequences were used as query sets to conduct local BLASTP searches against the reference genome assemblies of Gossypium hirsutum, Gossypium raimondii, Gossypium arboreum and Gossypium barbadense (https://yanglab.hzau.edu.cn/CottonMD, (accessed on 5 April 2025)), with a significance criterion of E-value < 1 × 10−5. The hidden Markov model (HMM) profile for the FBN domain (Pfam accession: PF04755) was retrieved from the Pfam database [29]. This profile was subsequently employed to screen protein databases using HMMER v3.0 (http://hmmer.org/, (accessed on 20 April 2025)), with a stringent E-value cutoff of <0.01 to ensure the reliability of the search results. The identities of the screened proteins were further verified by querying the SMART [30] and InterPro [31] databases. The identified cotton FBN genes were designated based on their sequence homology with Arabidopsis orthologs.

2.2. Multiple Alignment and Phylogenetic Analysis

Multiple sequence alignments and phylogenetic analyses were performed using MEGA 11.0 software. The Neighbor Joining (NJ) method was employed for phylogenetic tree reconstruction, with bootstrap support values calculated based on 1000 replicates to assess nodal confidence. All other algorithmic parameters were maintained at their default settings.

2.3. Structure Analysis and Cis-Element Prediction

Motif analysis was conducted using the MEME Suite v5.5.8 [32]. Promoter regions (2000 bp upstream of the translation start site) were extracted, and cis-regulatory elements were predicted using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, (accessed on 10 May 2025)). The gene structure and motif analysis results were visualized using TBtools software v2.225 [33].

2.4. Chromosomal Location and Collinearity Analysis

Chromosomal location information for genes was extracted from the genome GFF3 annotation data, and TBtools v2.225 software was employed to generate chromosomal mapping diagrams and conduct gene collinearity analysis [33].

2.5. In Vitro Ovule Culture and RNA-Seq

Ovules were harvested from +1 DPA upland cotton flowers. Following surface sterilization with 75% (w/v) ethanol, the ovules were dissected under sterile conditions and transferred to ovule in vitro culture medium [34]. For the experimental groups, culture media were prepared with final concentrations of 0.1 mM MeJA (methyl jasmonate), 0.5 mM GA3 (gibberellin A3), or 5 mM IAA (indole-3-acetic acid). All hormones were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Following independent cultivation at 30 °C for 10 days, total RNA was extracted from the collected samples, with 2 μg of RNA used for RNA-seq library construction. Transcriptome sequencing (RNA-seq) was performed on the Illumina NovaSeq 6000 platform, and subsequent data analysis was carried out by Wuhan IGENEBOOK Biotechnology Co. (Wuhan, China). Three independent biological replicates were included in the experiment.

2.6. Evolutionary Selection Pressure Analysis

Using the Easy Ka/Ks Calculator in TBtools v2.225 software [33], we calculated the nonsynonymous (Ka) and synonymous (Ks) substitution rates for duplicated FBN gene pairs that had been aligned by MEGA 11.0 software. According to the Ka/Ks ratio criteria (Ka/Ks > 1 indicating positive selection, Ka/Ks = 1 indicating neutral selection, and Ka/Ks < 1 indicating purifying selection [35]), we evaluated the selection pressure patterns for each gene pair.

2.7. Expression Pattern Analysis of Cotton FBN Genes

Transcriptome datasets from diverse tissues (ovules at 0 days post-anthesis (DPA), and fibers at 5, 10, 15, 20, and 25 DPA) [36] and hormone conditions (MeJA, GA3 and IAA treatments) were utilized. Gene expression levels were estimated as fragments per kilobase of exon per million reads (FPKM) using Cufflinks, normalized, and visualized via heatmaps generated with TBtools software [33].

2.8. qRT-PCR Analysis

Total RNA was extracted separately from ovules at 0 DPA and developing fiber tissues at 5, 10, 15, and 20 DPA of Jin668 using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotechnology, Nanjing, China). First-strand cDNA was synthesized from 1 μg of total RNA using the HiScript II Q RT SuperMix for qPCR (Vazyme Biotechnology) according to the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) was performed using the ChamQ SYBR qPCR Master Mix (Vazyme Biotechnology), with three biological replicates per gene. The cotton ubiquitin gene UBQ7 (GenBank accession: AY189972) was used as an internal reference, and primer sequences are listed in Table S1. Relative gene expression levels were calculated using the 2−ΔΔCT method [37].

3. Results

3.1. Identification of FBN Genes in Cotton

To identify FBN genes in cotton, we used 14 Arabidopsis FBN amino acid sequences as queries for BLASTP and HMMER3.0 searches, and identified 28 FBN proteins in G. hirsutum (Gh), 28 in G. barbadense (Gb), 13 in G. arboreum (Ga) and 15 in G. raimondii (Gr) (Table 1). Comparative genomic analysis revealed that allopolyploid cotton species (G. hirsutum and G. barbadense) possessed approximately twice the FBN gene count of diploid G. arboreum and G. raimondii (Table 1), indicating lineage-specific expansion of the FBN gene family during cotton polyploidization. In G. arboreum, the lengths of FBN genes ranged from 1031 bp to 5543 bp, encoding polypeptides composed of 215 to 677 amino acids. The predicted molecular weights spanned from 24.35 kDa to 76.67 kDa, with theoretical isoelectric points varying between 4.54 and 9.86 (Table 1). In G. raimondii, FBN gene lengths varied between 728 bp and 6103 bp, encoding polypeptides composed of 423 to 2031 amino acids. Predicted molecular weights spanned 15.87 kDa to 76.79 kDa, with theoretical isoelectric points ranging from 4.49 to 9.76 (Table 1). Similarly, in G. barbadense and G. hirsutum, notable variations were observed in the FBN gene attributes. Their gene lengths ranged from 609 bp to 12,587 bp, isoelectric points from 4.4 to 9.8, and molecular weights from 12.79 kDa to 76.68 kDa (Table 1). These findings suggest that the FBN gene family in cotton has undergone evolutionary expansion.

Table 1.

The FBN gene family members in cotton.

Gene Name Sequence ID Gene (bp) CDS (bp) MWa (kDa) Theoretical pI Species
GhFBN1A-1 Gh_D05G1665.1 3179 978 35.42335 5.14 G. hirsutum
GhFBN1A-2 Gh_A05G1494.1 1399 978 35.44943 5.14 G. hirsutum
GhFBN1B-1 Gh_D09G0662.1 5532 1008 36.86466 4.93 G. hirsutum
GhFBN1B-2 Gh_A09G0658.1 2380 1008 36.91571 5 G. hirsutum
GhFBN2-1 Gh_D09G1905.1 4196 1107 39.83073 4.49 G. hirsutum
GhFBN2-2 Gh_A09G1782.1 2257 1101 39.62955 4.58 G. hirsutum
GhFBN3-1 Gh_A01G1493.1 3987 735 27.42963 9.65 G. hirsutum
GhFBN3-2 Gh_D01G1730.1 3179 735 27.49574 9.65 G. hirsutum
GhFBN3-3 Gh_A04G1080.1 3987 762 28.53791 9.69 G. hirsutum
GhFBN3-4 Gh_D04G1687.1 3179 762 28.50085 9.54 G. hirsutum
GhFBN4-1 Gh_D09G2214.1 4196 831 29.92041 9.37 G. hirsutum
GhFBN4-2 Gh_A09G2222.1 3179 831 30.01153 9.37 G. hirsutum
GhFBN5-1 Gh_D06G1644.1 5532 1302 47.95253 6 G. hirsutum
GhFBN5-2 Gh_A06G1315.1 1399 1500 55.18532 9 G. hirsutum
GhFBN5-3 Gh_D11G3441.1 4196 429 15.74633 8.67 G. hirsutum
GhFBN6-1 Gh_D06G1142.1 5532 789 28.93921 9.4 G. hirsutum
GhFBN6-2 Gh_A06G0966.1 1399 789 28.98224 9.4 G. hirsutum
GhFBN6-3 Gh_D01G0744.1 3179 348 12.79046 8.06 G. hirsutum
GhFBN7A Gh_A09G1197.1 2380 912 33.32015 6.67 G. hirsutum
GhFBN7B Gh_D09G1203.1 4196 912 33.29012 6.67 G. hirsutum
GhFBN8-1 Gh_D11G1079.1 4196 714 26.46673 8.78 G. hirsutum
GhFBN8-2 Gh_A11G0937.1 3179 846 31.60553 9.22 G. hirsutum
GhFBN9-1 Gh_D07G1476.1 5532 651 24.49891 8.61 G. hirsutum
GhFBN9-2 Gh_A07G1372.1 1399 645 24.35177 8.61 G. hirsutum
GhFBN10-1 Gh_D08G0826.1 5532 1200 44.77003 6.48 G. hirsutum
GhFBN10-2 Gh_A08G0709.1 1399 1200 44.78029 8.64 G. hirsutum
GhFBN11-1 Gh_D12G2421.1 4196 2031 76.64194 9.74 G. hirsutum
GhFBN11-2 Gh_A12G2271.1 3179 2031 76.67501 9.86 G. hirsutum
GaFBN1A Ga05G1857.1 1399 978 35.43737 5.14 G. arboreum
GaFBN1B Ga09G0772.1 2379 1008 36.92974 5 G. arboreum
GaFBN2 Ga09G2235.1 1607 1101 39.6435 4.54 G. arboreum
GaFBN3A-1 Ga02G1224.1 2259 735 27.44765 9.65 G. arboreum
GaFBN3A-2 Ga04G0211.1 1736 762 28.54992 9.69 G. arboreum
GaFBN4 Ga09G2612.1 1590 831 29.95748 9.37 G. arboreum
GaFBN5 Ga11G1531.1 2064 867 32.03204 7.55 G. arboreum
GaFBN6 Ga06G1253.1 1113 705 25.7124 9.08 G. arboreum
GaFBN7A Ga09G1460.1 5543 912 33.29209 6.07 G. arboreum
GaFBN8 Ga11G2969.1 3024 846 31.58558 9.07 G. arboreum
GaFBN9 Ga07G1647.1 1031 645 24.35177 8.61 G. arboreum
GaFBN10 Ga08G0915.1 5511 1200 44.73919 8.65 G. arboreum
GaFBN11 Ga12G0290.1 4018 2031 76.67501 9.86 G. arboreum
GbFBN1A-1 Gbar_D05G017830.1 2891 978 35.40735 5.14 G. barbadense
GbFBN1A-2 Gbar_A05G017410.1 2838 978 35.44943 5.14 G. barbadense
GbFBN1A-3 Gbar_D09G007520.1 2745 1008 36.87478 4.99 G. barbadense
GbFBN1B Gbar_A09G007800.1 2780 1008 36.90267 4.95 G. barbadense
GbFBN2-1 Gbar_D09G020830.1 2524 1107 39.81765 4.49 G. barbadense
GbFBN2-2 Gbar_A09G021110.1 2508 1101 39.65757 4.58 G. barbadense
GbFBN3A-1 Gbar_D01G018650.1 5258 747 28.08431 9.34 G. barbadense
GbFBN3A-2 Gbar_A01G017480.1 2517 735 27.42963 9.65 G. barbadense
GbFBN3A-4 Gbar_A04G013910.1 1706 762 28.53392 9.69 G. barbadense
GbFBN3A-3 Gbar_D04G018570.1 1864 762 28.49887 9.54 G. barbadense
GbFBN4-1 Gbar_D09G024190.1 2000 831 29.91642 9.37 G. barbadense
GbFBN4-2 Gbar_A09G024530.1 1568 813 29.46222 8.77 G. barbadense
GbFBN5-1 Gbar_D06G016800.1 3442 1614 59.91259 7.03 G. barbadense
GbFBN5-2 Gbar_A06G016120.1 2029 897 33.38748 7.64 G. barbadense
GbFBN5-3 Gbar_D11G024180.1 12587 894 33.03026 9.27 G. barbadense
GbFBN5-4 Gbar_A11G023440.1 2514 552 20.34894 9.43 G. barbadense
GbFBN6-1 Gbar_D06G011790.1 1462 705 25.69841 9.19 G. barbadense
GbFBN6-2 Gbar_A06G011340.1 1453 717 26.18494 8.94 G. barbadense
GbFBN7B-1 Gbar_D09G013520.1 6058 912 33.29012 6.67 G. barbadense
GbFBN7A-2 Gbar_A09G013680.1 5971 912 33.32015 6.67 G. barbadense
GbFBN8-1 Gbar_D11G011270.1 3392 846 31.58953 9.25 G. barbadense
GbFBN8-2 Gbar_A11G010770.1 3987 1014 37.98029 9.67 G. barbadense
GbFBN9-1 Gbar_D07G016160.1 3560 651 24.49891 8.61 G. barbadense
GbFBN9-2 Gbar_A07G015770.1 3386 645 24.35177 8.61 G. barbadense
GbFBN10-1 Gbar_D08G008730.1 5745 954 35.6023 6.46 G. barbadense
GbFBN10-2 Gbar_A08G008420.1 5752 1119 41.6738 7.7 G. barbadense
GbFBN11-1 Gbar_D12G026080.1 4751 1425 54.09186 9.87 G. barbadense
GbFBN11-2 Gbar_A12G026140.1 4788 2031 76.68903 9.86 G. barbadense
GrFBN1A-1 Gorai.009G182500.1 2513 978 35.42335 5.14 G. raimondii
GrFBN1A-2 Gorai.006G082400.1 2754 1014 37.13302 5.06 G. raimondii
GrFBN2 Gorai.006G217700.1 2488 1125 40.3022 4.49 G. raimondii
GrFBN3A-1 Gorai.002G209300.1 2559 735 27.42461 9.56 G. raimondii
GrFBN3A-2 Gorai.012G160700.1 1982 762 28.48682 9.54 G. raimondii
GrFBN4 Gorai.006G252600.1 2015 831 29.97849 9.37 G. raimondii
GrFBN5-1 Gorai.010G181700.1 3037 1812 67.68879 8.5 G. raimondii
GrFBN5-2 Gorai.002G151300.1 1994 813 30.16 9.12 G. raimondii
GrFBN6-1 Gorai.010G123700.1 1498 705 25.68439 9.19 G. raimondii
GrFBN6-2 Gorai.002G101300.1 728 423 15.86637 9.16 G. raimondii
GrFBN7B Gorai.006G142800.1 6103 912 33.29012 6.67 G. raimondii
GrFBN8 Gorai.007G115100.1 3493 861 32.16422 9.36 G. raimondii
GrFBN9 Gorai.001G175700.1 3596 651 24.5059 8.61 G. raimondii
GrFBN10 Gorai.004G092800.1 5850 1200 44.80911 6.93 G. raimondii
GrFBN11 Gorai.008G269500.1 4726 2031 76.79095 9.76 G. raimondii
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3.2. Phylogenetic Relationships of FBN Genes

To clarify the evolutionary relationships of the FBN gene family in cotton, we constructed a phylogenetic tree using the Neighbor Joining (NJ) method with the FBN protein sequences from A. thaliana, G. arboreum, G. raimondii, G. hirsutum and G. barbadense. Cotton FBN genes from G. arboreum, G. raimondii, G. hirsutum and G. barbadense are named by their phylogenetic positions relative to AtFBN genes. Phylogenetic analysis revealed that FBNs are classified into 11 groups (Figure 1). The groups FBN2, FBN4, FBN8, FBN9, FBN10, and FBN11 exhibited a highly conserved evolutionary pattern. These gene groups experienced independent expansions in G. arboreum, G. raimondii, G. hirsutum, and G. barbadense, and manifested a one-to-one orthologous relationship. During evolution in G. arboreum, the genes GaFBN3B and GaFBN7B were lost, whereas G. raimondii underwent losses of GrFBN1B, GrFBN3B, and GrFBN7A. The hybridization events in allotetraploid cotton species (G. hirsutum and G. barbadense) might have driven this unbalanced evolutionary trajectory.

Figure 1.

Figure 1

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The phylogenetic tree of the FBN family in cotton. Phylogenetic analysis, performed using MEGA11.0, included Arabidopsis (14), G. hirsutum (28), G. barbadense (28), G. arboreum (13) and G. raimondii (15) sequences. Different subfamilies are color-coded for clear differentiation.

3.3. Structure Analysis of Cotton GhFBN Genes

To comprehensively explore the structural diversity of FBN genes, we conducted an analysis of the sequence structures, motifs, and conserved domains of the identified FBN genes in G. hirsutum, guided by phylogenetic analysis (Figure 2A). With the exceptions of GhFBN6-3 and GhFBN5-1, the remaining genes shared similar motifs (Figure 2B). All members of the FBN family possess the PAP-fibrillin domain, a characteristic structural feature that defines this gene family (Figure 2C). Moreover, the exon/intron structures of FBN genes within the same group were strikingly alike compared to those in other groups (Figure 2D). These findings imply that genes with unique structural features might perform specialized biological functions.

Figure 2.

Figure 2

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The structural distribution patterns of the 28 GhFBN genes. (A) Construction of a phylogenetic tree using GhFBN protein sequences. (B) The distribution of MEME conserved motifs, where different conserved motifs are indicated by colored boxes. (C) The distribution of the PAP fibrillin conserved domain, with the green square signifying this conserved domain. (D) The gene structure of GhFBN, in which exons are represented by blue boxes.

3.4. Chromosomal Location and Gene Duplication of GhFBN Genes

To investigate the chromosomal distribution of GhFBN genes, each GhFBN gene was mapped to its corresponding chromosome using gene information from the NBI database. For a deeper exploration of GhFBN gene evolution across four cotton species, genome duplication events including whole-genome duplication (WGD), segmental duplication, and tandem duplication were examined. In G. hirsutum, 28 GhFBN genes display a heterogeneous distribution across the 18 chromosomes. Specifically, there are 13 GhFBN genes in the A chromosome group and 15 GhFBN genes in the D chromosome group (Figure 3). Chromosome D09 harbors the highest number of GhFBN genes, with four. In contrast, GhFBN4-2 and GhFBN5-3 are mapped to scaffolds. To decipher the expansion pattern of the GhFBN gene family in G. hirsutum, Circos analysis was carried out. The results demonstrate that 26 GhFBN family genes have experienced tandem duplication and are dispersed across chromosomes A01, A04, A05, A06, A07, A08, A09, A11, D01, D04, D05, D06, D07, D08, D09 and D11 (Figure 4). The remaining two genes are scattered within the G. hirsutum genome. We further delved into the FBN gene family in G. hirsutum, G. arboreum, G. raimondii and G. barbadense, and explored the gene duplication patterns of FBN genes in G. arboreum and G. barbadense (Figure 5). The duplicate gene pairs of these four cotton species reveal the basis for polyploidization and large-scale expansion of the FBN gene family during the evolutionary process.

Figure 3.

Figure 3

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The chromosomal locations of FBN in the Gossypium hirsutum species. Gene IDs are annotated on the right, whereas vertical ideograms on the left depict both gene loci and chromosomal lengths. Mb, megabase.

Figure 4.

Figure 4

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The distribution and duplication of GhFBN genes in G. hirsutum. The distribution of GhFBN genes across the 24 chromosomes of the G. hirsutum genome. Duplicated genes are connected by red lines.

Figure 5.

Figure 5

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The collinearity of FBN genes among various cotton species. The chromosomes of G. raimondii, G. hirsutum, G. arboreum and G. barbadense are shown in different colors, marked in orange, purple, green, and pink, respectively. The blue lines indicate the collinear relationships among FBN genes.

3.5. Selection Pressure Ka/Ks Analysis

To elucidate the selective pressures acting on GhFBN genes during gene duplication in upland cotton, this study comprehensively analyzed coding sequences of all intragenomic paralogous pairs. Homologous coding regions free of frameshift mutations were identified through sequence alignment, followed by computation of nonsynonymous (Ka) and synonymous (Ks) substitution rates for each paralog pair using TBtools. Evolutionary selective pressures were inferred based on Ka/Ks ratio analyses. In G. hirsutum GhFBNs, 17 gene pairs exhibited Ka/Ks < 0.5 and 3 displayed ratios of 0.5–1, with strong purifying selection and high sequence conservation inferred in the majority and near-neutral evolution detected in the remainder (Table 2). These findings suggest that functional divergence was constrained by dosage effects or evolutionary time constraints post-whole-genome/segmental duplication.

Table 2.

Ka/Ks analysis of GhFBN gene families in Gossypium hirsutum.

Sequence ID Ka Ks Ka/Ks
GhFBN3-1&GhFBN3-3 0.25646808027089535 1.8899984771994518 0.1356975063021864
GhFBN3-1&GhFBN3-2 0.015378195927205629 0.031811997515935844 0.48340868628265493
GhFBN3-1&GhFBN3-4 0.26519839929406197 2.0183731848080146 0.1313921534878533
GhFBN3-3&GhFBN3-2 0.2539649643874308 1.9922119769228535 0.12747888644847022
GhFBN3-3&GhFBN3-4 0.010415327224946307 0.022316848711233652 0.4667024166231648
GhFBN1A-2&GhFBN1B-2 0.11398702336283616 0.6464385028816231 0.17633080773301285
GhFBN1A-2&GhFBN1A-1 0.002682766106657679 0.026415055967744308 0.10156200728605806
GhFBN1A-2&GhFBN1B-1 0.11237961274006769 0.6372917984117465 0.17633933626658815
GhFBN6-2&GhFBN6-1 0.013447621783443438 0.03803440541457851 0.35356466433123185
GhFBN5-2&GhFBN5-1 0.037268406863882296 0.05082235814271873 0.7333073124868705
GhFBN9-2&GhFBN9-1 0.006157038170299509 0.0812977842242655 0.07573439090682837
GhFBN10-2&GhFBN10-1 0.014196438052508897 0.025840759627618143 0.5493816070846461
GhFBN1B-2&GhFBN1A-1 0.11091080011024723 0.6246818422013267 0.17754766125329788
GhFBN1B-2&GhFBN1B-1 0.014403061291802292 0.0434431959190317 0.33153779290654267
GhFBN7A&GhFBN7B 0.004380640343959717 0.04578080007805861 0.0956872823648888
GhFBN2-2&GhFBN2-1 0.009753261495566346 0.04104225646267837 0.23763950465139363
GhFBN8-2&GhFBN8-1 0.03976139397807425 0.04769566096457199 0.8336480336777121
GhFBN11-2&GhFBN11-1 0.008441190776946137 0.02530551062085989 0.3335712487061169
GhFBN3-2&GhFBN3-4 0.2626554249946424 2.1422101503898068 0.12260955114363936
GhFBN1A-1&GhFBN1B-1 0.11243405990473135 0.6358361961198246 0.17682865585642585
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3.6. Analysis of Putative Cis-Acting Elements in GhFBN Promoters

To further explore the biological activities of GhFBNs in G. hirsutum, we obtained the 2 kb upstream promoter regions of all GhFBN genes and analyzed their cis-acting regulatory elements with the PlantCARE database. In the promoter regions of GhFBN genes, we identified multiple types of regulatory elements (Figure 6). These elements included those responsive to plant hormones, such as ABRE (abscisic acid), CGTCA-motif and TGACG-motif (methyl jasmonate), P-box, TATC-box, and GARE-motif (gibberellic acid), AuxRR-core and TGA-element (auxin), and TCA-element (salicylic acid), indicating that the expression of GhFBNs is regulated by different plant hormones (Table 3). Moreover, light-responsive elements like L-box were also detected. Overall, these results suggest that GhFBNs participate in plant growth, development, and responses to environmental stresses.

Figure 6.

Figure 6

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Analysis of cis-acting elements in the upstream region of the GhFBN gene promoter. Boxes in different colors represent the uniquely identified cis-acting elements.

Table 3.

Analysis of cis-acting elements in GhFBN.

ID Cis-Elements Name Functions of Cis-Elements
Gh_A01G1493 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A01G1493 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A04G1080 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A04G1080 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_A04G1080 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A04G1080 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A04G1080 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A04G1080 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A04G1080 AuxRR-core cis-acting regulatory element involved in auxin responsiveness
Gh_A05G1494 P-box gibberellin-responsive element
Gh_A05G1494 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A05G1494 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A05G1494 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A05G1494 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A05G1494 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A05G1494 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A05G1494 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A05G1494 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_A05G1494 GARE-motif gibberellin-responsive element
Gh_A06G0966 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A06G0966 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_A06G0966 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A06G0966 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A06G0966 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A06G0966 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A06G0966 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A06G1315 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_A06G1315 AuxRR-core cis-acting regulatory element involved in auxin responsiveness
Gh_A06G1315 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A07G1372 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A07G1372 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A07G1372 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_A08G0709 GARE-motif gibberellin-responsive element
Gh_A09G0658 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A09G0658 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A09G0658 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_A09G1197 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_A09G1197 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_A09G1782 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A09G1782 AuxRR-core cis-acting regulatory element involved in auxin responsiveness
Gh_A09G1782 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A09G1782 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A09G2222 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A09G2222 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A09G2222 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A09G2222 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_A09G2222 P-box gibberellin-responsive element
Gh_A09G2222 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A11G0937 GARE-motif gibberellin-responsive element
Gh_A11G0937 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A11G0937 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A11G0937 TGA-element auxin-responsive element
Gh_A11G0937 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_A11G0937 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_A11G0937 P-box gibberellin-responsive element
Gh_A12G2271 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_A12G2271 GARE-motif gibberellin-responsive element
Gh_A12G2271 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A12G2271 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A12G2271 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A12G2271 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A12G2271 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A12G2271 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A12G2271 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A12G2271 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_A12G2271 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A12G2271 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A12G2271 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A12G2271 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A12G2271 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_A12G2271 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D01G0744 TGA-element auxin-responsive element
Gh_D01G0744 P-box gibberellin-responsive element
Gh_D01G0744 GARE-motif gibberellin-responsive element
Gh_D01G0744 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_D01G0744 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_D01G0744 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D01G1730 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D01G1730 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D01G1730 AuxRR-core cis-acting regulatory element involved in auxin responsiveness
Gh_D04G1687 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D04G1687 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D04G1687 AuxRR-core cis-acting regulatory element involved in auxin responsiveness
Gh_D04G1687 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D04G1687 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D04G1687 P-box gibberellin-responsive element
Gh_D04G1687 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_D05G1665 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_D05G1665 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_D05G1665 GARE-motif gibberellin-responsive element
Gh_D05G1665 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D05G1665 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D05G1665 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D05G1665 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D05G1665 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D05G1665 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D05G1665 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D05G1665 P-box gibberellin-responsive element
Gh_D05G1665 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_D06G1142 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D06G1142 AuxRR-core cis-acting regulatory element involved in auxin responsiveness
Gh_D06G1142 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D06G1142 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D06G1142 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D06G1142 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D06G1142 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D06G1142 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_D06G1142 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D06G1644 P-box gibberellin-responsive element
Gh_D06G1644 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D06G1644 TGA-element auxin-responsive element
Gh_D06G1644 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D06G1644 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D06G1644 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D06G1644 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_D07G1476 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D07G1476 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D07G1476 GARE-motif gibberellin-responsive element
Gh_D07G1476 TGA-element auxin-responsive element
Gh_D08G0826 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D08G0826 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D08G0826 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G0662 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G0662 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_D09G1203 P-box gibberellin-responsive element
Gh_D09G1203 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D09G1203 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G1203 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G1203 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D09G1203 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_D09G2214 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G2214 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G2214 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G2214 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G2214 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G2214 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G2214 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D09G2214 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D09G2214 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D09G2214 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D09G2214 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D09G2214 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D11G1079 P-box gibberellin-responsive element
Gh_D11G1079 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_D11G1079 TATC-box cis-acting element involved in gibberellin responsiveness
Gh_D11G1079 TGA-element auxin-responsive element
Gh_D11G1079 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D11G1079 GARE-motif gibberellin-responsive element
Gh_D11G3441 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D11G3441 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D11G3441 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D11G3441 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D12G2421 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D12G2421 CGTCA-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D12G2421 GARE-motif gibberellin-responsive element
Gh_D12G2421 TCA-element cis-acting element involved in salicylic acid responsiveness
Gh_D12G2421 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D12G2421 TGACG-motif cis-acting regulatory element involved in MeJA responsiveness
Gh_D12G2421 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D12G2421 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D12G2421 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D12G2421 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D12G2421 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D12G2421 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D12G2421 ABRE cis-acting element involved in abscisic acid responsiveness
Gh_D09G1905 L-box part of a light-responsive element
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3.7. Expression Profiling Based on Transcriptome Data

To delve deeper into the functions of GhFBN genes in cotton, we analyzed RNA sequencing data from various tissues of upland cotton, including seeds, roots, stems, flowers, leaves, 0 DPA ovules, and fibers at different developmental stages. The results showed that GhFBN genes were predominantly expressed in flowers, with leaves exhibiting the second-highest expression levels (Figure 7A). Genes including GhFBN2-1, GhFBN2-2, GhFBN3-3, GhFBN7A, and GhFBN7B showed elevated expression at the fiber initiation stage (Figure 7B). Significantly, GhFBN1A-1, GhFBN1A-2 and GhFBN2-2 were more highly expressed during the fiber elongation stage (Figure 7B). Notably, GhFBN2-2 maintained consistently high expression throughout the entire fiber development process (Figure 7B). To further explore the evolutionary basis of this expression pattern, a comparative analysis of A- and D-subgenome divergence revealed distinct transcriptional dynamics among homologous genes during fiber development. Specifically, while GhFBN2-2 (A-subgenome homoeolog) exhibits sustained high expression, its D-subgenome counterpart GhFBN2-1 shows significantly reduced transcription during critical fiber developmental stages, suggesting minimal functional involvement in this process. In contrast, the robust, consistent expression of GhFBN2-2 across fiber development indicates potential adaptive specialization in regulating spatiotemporal fiber developmental programs. Based on the transcriptomic expression data from different fiber development stages, we performed targeted validation of GhFBN1A-1, GhFBN1A-2, GhFBN2-1, GhFBN2-2, GhFBN7A, and GhFBN7B via quantitative real-time PCR (qRT-PCR), and the results were consistent with the expression trends observed in the transcriptomic data (Figure 7C). Collectively, these findings strongly suggest that GhFBNs play pivotal roles in fiber development.

Figure 7.

Figure 7

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Expression analysis of GhFBN genes in different cotton tissues. (A) Expression of GhFBNs in seeds, roots, stems, petals and leaves. (B) Expression of GhFBNs in fibers at different developmental stages. The heatmap shows the Log2 (FPKM+1) of GhFBNs. Blue represents low expression, while red indicates high expression. DPA, days post-anthesis. (C) Expression of GhFBN genes in cotton fibers. Expression of GhFBNs is normalized based on the expression of GhUBQ7. Error bars represent ±SD (n = 3).

Promoter analysis predicted potential involvement of GhFBNs in phytohormone signaling pathways. Previous studies have demonstrated that GA (gibberellin acid) and IAA can promote fiber development, whereas JA (jasmonic acid) inhibits fiber development [34,38,39]. To validate this hypothesis, transcriptomic sequencing was performed on 10 DPA fibers derived from in vitro-cultured ovules, with experimental groups treated with MeJA, GA3, or IAA and non-hormonally treated samples included as the control (CK). Results revealed that GhFBN1A-2 expression was significantly downregulated by MeJA but strongly induced by GA3, suggesting it may have a positive regulatory role in GA3-mediated fiber development (Figure 8). In contrast, GhFBN5-3 and GhFBN6-3 are hardly expressed in fibers before and after GA3 and IAA treatments, indicating that they may not be involved in GA3- and IAA-induced fiber elongation (Figure 8B,C). Meanwhile, the expression level of GhFBN11-2 in fiber cells was downregulated following GA3 treatment, while that of GhFBN3-3 in fiber cells was similarly reduced after IAA treatment. These findings indicate that these genes exhibit a negative correlation with fiber elongation mediated by gibberellin or auxin, respectively (Figure 8B,C). Building on these transcriptomic insights, we performed qRT-PCR validation on GhFBN1A-2, GhFBN11-2, and GhFBN3-3 under identical hormonal treatments, confirming consistent expression trends with the transcriptomic data. Collectively, these data highlight the diverse regulatory roles of GhFBNs in phytohormone-mediated cotton fiber elongation.

Figure 8.

Figure 8

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Expression profiling of GhFBN genes in cotton fibers at 10 DPA upon hormone treatment. (A) Expression of GhFBNs in fibers treated with MeJA. (B) Expression of GhFBNs in fibers treated with GA3. (C) Expression of GhFBNs in fibers treated with IAA. The heatmap shows the Log2 (FPKM+1) of GhFBNs. Blue represents low expression, while red indicates high expression. CK, control. MeJA, methyl jasmonate. GA3, gibberellin A3. IAA, indole-3-acetic acid. (D) Expression of GhFBN genes in cotton ovules cultured in vitro, with and without hormone treatment. Expression of GhFBNs is normalized based on the expression of GhUBQ7. Error bars represent ±SD (n = 3). Asterisks indicate significant differences by t-test; ** p ≤ 0.01; *** p ≤ 0.001.

4. Discussion

The FBN gene family has been identified on a whole-genome scale in numerous plant species, and considerable insights have been gained into its potential functions in the growth and development of plants such as pepper and potato [10,20,40]. Cotton, being a crucial economic crop, holds significant agricultural and industrial value. Remarkably, however, there has been no report on the whole-genome study of the FBN gene family in upland cotton (Gossypium hirsutum). Moreover, whether FBN genes are involved in the growth and development of cotton and, if so, what roles they play remain largely unknown, thus presenting an important area for further exploration.

From an evolutionary perspective, the number of FBN genes varies among different cotton species. For example, 28, 28, 13 and 15 members of the FBN gene family were identified in G. hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. This, together with the situations of the FBN gene family in other plants, indicates that the FBN gene family has undergone expansion and differentiation during the plant evolution process. The number of FBN genes in allotetraploid cotton species is approximately twice that in diploid G. arboreum and G. raimondii, suggesting a lineage-specific expansion of the FBN gene family during cotton polyploidization. This expansion likely provides a more abundant genetic basis for cotton to adapt to diverse environments and meet its own developmental needs.

In terms of functions, FBN genes play crucial roles in plant growth, development, and stress responses [41]. In cotton, different GhFBN genes exhibit specific expression patterns at various stages of fiber development. For instance, GhFBN7A, GhFBN7B, and other genes show high expression levels at the fiber initiation stage, while GhFBN1A-1 and GhFBN2-2 are more highly expressed during the fiber elongation stage. The sustained high expression of GhFBN2-2 throughout fiber development, coupled with its distinct transcriptional divergence from the D-subgenome homoeolog GhFBN2-1, which shows significantly reduced transcription at critical stages, highlights subgenome-specific functional differentiation in cotton fiber traits. This expression pattern strongly supports the adaptive specialization of the A-subgenome GhFBN2-2 in regulating the spatiotemporal programs underlying fiber development, emphasizing its potential role in this agronomically vital process. This is consistent with the functions of FBN genes in the development processes of other plants. For example, in tomatoes, most SlFBNs play important roles in leaf development [22]. This indicates that FBN genes have conserved functions in specific tissues and developmental stages of different plants.

The differential expression of GhFBNs in response to MeJA, GA3, and IAA treatments reveals their specialized roles in hormone-mediated cotton fiber elongation. Strong induction of GhFBN1A-2 by GA and simultaneous repression by JA highlight its potential as a positive regulator in GA-driven fiber development, while GA-induced downregulation of GhFBN11-2 and IAA-induced downregulation of GhFBN3-3 suggest their negative regulatory roles in hormone-induced growth. Notably, GhFBN5-3 and GhFBN6-3 show negligible expression changes, indicating a lack of involvement in these processes. These findings validate the involvement of GhFBNs in phytohormone signaling and provide critical insights for targeted molecular breeding to improve cotton fiber quality.

Regarding the response to abiotic stresses, existing studies have demonstrated that the FBN gene family is involved in plant responses to stresses such as drought [16,42,43]. Although there is currently no direct evidence of the specific association between GhFBN genes and drought stress in cotton, based on research on other plants, it can be inferred that GhFBN genes may also be involved. For example, in potatoes, drought stress increases the mRNA and protein abundances of CDSP 34 (FBN1) [44,45]. In wheat, the expressions of multiple FBN genes, such as TaFBNA1, are significantly upregulated under drought, low-temperature, and other stress conditions [14]. Therefore, GhFBN genes in cotton are likely to also play important roles in responses to drought and other abiotic stresses, which is worthy of further investigation.

In addition, the interactions between FBN proteins also affect the exertion of their functions [46]. In cotton, there is currently little research on the interactions of FBN proteins, so we refer to studies on other plants, such as the interaction between FBN1a and FBN2, and the interactions between FBN5 and SPS1, and between FBN5 and SPS2, respectively, in Arabidopsis [15,47]. FBN1A and FBN1B may form homodimers, heterodimers, or oligomers to interact and exert their functions [48]. It can be speculated that there is a complex interaction network among FBN proteins in cotton. These interactions may jointly regulate the growth, development, and stress response processes of cotton.

The FBN gene family in cotton shares similarities and specificities with those in other plants in terms of evolution, functions, and protein–protein interactions, and this study further reveals their potential in fiber quality improvement through functional characterization. Key targets are identified: leveraging GhFBN2-2’s consistent high expression during fiber development, enhancing GhFBN1A-2 GA3-induced growth promotion, and avoiding GhFBN11-2 and GhFBN3-3 negative interference with hormone signaling. Future research can further focus on the specific mechanisms of cotton FBN genes in stress responses and their interaction relationships. This will not only contribute to a deeper exploration of the regulatory mechanisms of cotton growth and development but also provide a solid theoretical basis for the genetic improvement of cotton.

5. Conclusions

In conclusion, we identified 28, 28, 13 and 15 members of the FBN gene family in G. hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. This study provides the first comprehensive characterization of FBN genes in cotton and establishes the role of GhFBN1A-2 in fiber development. By integrating analyses of gene structure, evolutionary dynamics, expression patterns, and functional validation, we delineated the evolutionary trajectories and functional diversification of FBN genes during cotton genome evolution. These findings not only deepen our understanding of the biological roles of FBNs in fiber development but also provide a foundation for future genetic improvement of cotton fiber quality. These findings deepen our understanding of GhFBN-mediated regulation of cotton fiber development stages like elongation and cell wall formation, provide a molecular basis for genetic improvement of fiber quality, and identify target genes to guide precision breeding for enhanced traits such as length and strength through clarified functional characteristics and regulatory patterns of specific GhFBN members in hormone-mediated growth.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/biology14081012/s1. Table S1: The primers used in this study.

biology-14-01012-s001.zip (9.6KB, zip)

Author Contributions

L.L. and G.X. conceived the research. S.Y. and L.H. carried out the experiments. Z.F. and L.Z. analyzed the data. L.Z., L.L. and G.X. prepared the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that supports this study are available in the Supplementary Materials of this article.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.

Footnotes

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Supplementary Materials

biology-14-01012-s001.zip (9.6KB, zip)

Data Availability Statement

The data that supports this study are available in the Supplementary Materials of this article.

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