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Article

Genome-Wide Identification and Expression Analysis of the GRF Gene Family in Gossypium hirsutum L.

by
Cong-Hua Feng
1,†,
Linlin Liu
1,†,
Di Liu
1,
Junbo Zhen
1,
Mengzhe Li
1,
Mengmeng Jiang
1,2 and
Jina Chi
1,*
1
Institute of Cotton, Hebei Academy of Agricultural and Forestry Sciences/Key Laboratory of Biology and Genetic Improvement of Cotton in Huanghuaihai Semiarid Area, Ministry of Agriculture and Rural Affairs, Hebei Key Laboratory of Cotton Bio-Breeding and Cultivation Physiology, Shijiazhuang 050051, China
2
Genetics Laboratory, College of Life Science, Hebei University, Baoding 071002, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2026, 27(5), 2191; https://doi.org/10.3390/ijms27052191
Submission received: 27 January 2026 / Revised: 13 February 2026 / Accepted: 21 February 2026 / Published: 26 February 2026
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

Growth Regulating Factors (GRFs) are plant-specific transcription factors that play crucial roles in regulating growth and development throughout the plant life cycle. A total of 34 Gossypium hirsutum GRF family genes were identified at the genome-wide level, which were unevenly distributed on 19 chromosomes, and were predicted to be mainly localized in the nucleus and plasma membrane. The number of GRF family genes varied greatly among different species, and they were categorized into four subfamilies (I–IV) according to their phylogenetic relationships. The G. hirsutum GRF genes possessed specific highly conserved structural domains, Trp-Arg-Cys motif (WRC) and Gln, Leu, Gln motif (QLQ), and structural analysis of the genes revealed that they contained 1–23 exons, and most of them contained UTRs. Intraspecies covariance analysis revealed that the GRF genes expanded in G. hirsutum by segmental duplication. The promoter region of the G. hirsutum GRF gene contained a large number of adversity stress response elements, as well as a small number of hormone response elements and growth and development-related response elements. Transcriptome data showed that the expression of G. hirsutum GRF genes was significantly higher in leaves than in other tissues, and some GRF genes responded to a variety of abiotic stresses. Additionally, transcriptomic sequencing revealed significantly higher expression levels of GhGRFs (e.g., GhGRF13/14/18) in embryonic callus (EC) compared to non-embryonic callus (NEC). This differential expression was validated by RT-qPCR, which confirmed that GhGRF13/14/16/20 were significantly upregulated in EC relative to NEC. These findings provide valuable candidate genes and molecular insights for improving G. hirsutum regeneration efficiency and yield-related traits through genetic manipulation, thereby accelerating the molecular breeding of elite G. hirsutum varieties.

1. Introduction

As a primary global source of natural fiber, cotton is a vital cash crop and a fundamental raw material for the textile industry [1,2,3]. However, cotton production faces a number of challenges, including attacks by pests and diseases, increasing environmental pressures, and bottlenecks in genetic improvement [4]. In order to meet these challenges, the development of cotton breeding technology is particularly important. Among various breeding approaches, the induction and utilization of embryogenic callus (EC) in cotton have garnered significant attention due to its unique biological characteristics and application potential [5,6]. Cotton somatic embryogenesis plays a crucial role in the genetic transformation of cotton [7,8]. Embryogenic callus refers to a proliferative mass of cells derived from induced embryonic or embryogenic tissues, possessing regenerative capacity [9,10]. This type of callus not only retains embryogenic potential but can also proliferate indefinitely under appropriate culture conditions, thereby providing essential experimental material for genetic transformation, variety improvement, and biotechnological research in cotton [11,12,13,14]. Through embryogenic callus, efficient genetic transformation can be achieved, facilitating the selection of novel cultivars with desirable traits. In addition, embryonic healing tissues play a key role in the study of developmental biology, cell biology and molecular biology of cotton, which helps to reveal the molecular mechanism of cotton growth and development [6]. During cotton development, a large number of genes are differentially expressed and interact with each other at different stages and in different tissues, and this complex regulatory mechanism ensures the normal development of somatic embryos [7]. Although the induction and application of embryonic healing tissues in cotton are of great significance in cotton breeding and biotechnology research, there are still some technical and theoretical problems that need to be solved.
GRFs typically contain the Trp-Arg-Cys motif (WRC) and Gln, Leu, Gln motif (QLQ) structural domains [15,16,17]. In addition, three less-conserved structural domains (TQL, FFD, GGPL) have been identified in the C-terminal region of GRF proteins; however, the specific functions of these less-conserved motifs remain unclear [18,19]. GRFs have been extensively studied, and their roles in plant-healing tissue formation have been initially explored [20,21]. Lu et al. previously identified a novel rice gene, growth regulatory factor 1 (OsGRF1), which encodes a suspected transcription factor that plays a regulatory role in stem elongation [22,23]. A large number of GRF genes have been identified in Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, and Triticum aestivum [24,25,26]. GRFs function to regulate leaf growth and act as positive regulators of cell proliferation, affecting leaf shape and size [27,28]. PpnGRF5-1 is overexpressed in poplar and results in larger leaves than the wild type by increasing cell division and cell expansion [29,30]. Overexpression of BnGRF2a resulted in 20% leaf elongation in kale-type oilseed rape, and overexpression of AtGRF1 resulted in larger leaves [19,31]. GRF mutant leaves were smaller, and AtGRF9 negatively regulated leaf development by inhibiting A. thaliana leaf primordial cell proliferation [21,32,33,34,35].
This study was designed to characterize the induction mechanisms and genetic stability of cotton embryogenic tissues, with the ultimate goal of facilitating their application in cotton breeding. This was done by optimizing the induction and cultivation conditions of guiding tissues to improve their regeneration efficiency and genetic stability; by using modern molecular biology techniques to analyze the mechanism of guiding tissues in cotton breeding; and by exploring new strategies to combine guiding tissue technology with gene editing and marker-assisted selection, etc., with a view to providing new theoretical and technical support for the genetic improvement and varietal innovation of cotton. The results of this study will not only provide new theories and methods for cotton breeding, but also provide reference for biotechnology research of other crops.

2. Results

2.1. Genome-Wide Identification and Evolutionary Analysis of GhGRF Genes in Terrestrial G. hirsutum

In the whole genome data of G. hirsutum, 34 G. hirsutum GRF genes were finally screened by BLAST (Table S1). The protein database analysis in the NCBI database determined that all of them contained WRC and QLQ structural domains and belonged to the GRF gene family. The genes were named according to the order of the corresponding chromosome positions (GhGRF1- GhGRF34). The GhGRF genes were distributed on 19 chromosomes of the G. hirsutum genome (Figure S1), with three GhGRF genes on five chromosomes, two GhGRF genes on another five chromosomes, and one GhGRF gene on the remaining nine chromosomes. Next, analysis of the physicochemical properties of the proteins of the GhGRFs gene family revealed that the GhGRF genes encoded 182–610 amino acids, and the molecular weights of the proteins were relatively large, all of which were greater than 20 kDa (Table S1). The theoretical pIs of the GhGRFs gene family ranged from 5.89 to 9.65, with eight genes being acidic proteins and 26 genes being basic proteins. All 34 members of the GhGRFs gene family are stable with an instability index greater than 40. All GhGRF proteins are hydrophilic. Most of the GhGRF proteins are located in the nucleus and three genes are located in the plasma membrane (GhGRF8/13/30) as predicted by the Plant-mPLoc database.
Phylogenetic tree analysis classified the G. hirsutum GRF genes into four subfamilies, and the genes belonging to the same subfamily were closely related and highly homologous (Figure 1A). These results suggest that the GRFs gene family is highly conserved throughout evolution. We analyzed the number of GRF genes in different species and found that there were significant differences in the number of GRF genes in different species (Figure 1B). There was only one GRF gene in Marchantia polymorpha, while Salix purpurea possessed 43 GRF genes.

2.2. Motif, Conserved Structural Domain and Gene Structure Analysis of the GhGRF Genes

All 34 members of the GhGRFs gene family possess Motif2, Motif3, and Motif1, and their sequences are very conserved (Table S2). All GhGRF genes contain the WRC and QLQ structural domains, and GhGRF25 also contains the RETICULATA-like super family conserved structural domains. Gene structure analysis of the GhGRFs gene family revealed that they contain 1–23 exons, 0–23 introns, and 0–4 UTRs. GhGRF21 contains the highest number of 23 exons and 23 introns, and GhGRF10 and GhGRF32 do not contain UTRs (Figure 2).

2.3. Collinearity Analysis of GhGRF Genes

During the evolutionary process, the G. hirsutum GhGRFs gene family has undergone several duplication events. As shown in Figure 3, 34 GhGRF genes within the G. hirsutum genome formed 57 gene pairs by chromosome segmental duplication (SD). Then, we plotted the covariance analysis of the G. hirsutum GhGRFs gene family with five other plants (A. thaliana, O. sativa, T. aestivum, P. trichocarpa, and Zea mays) (Figure 4). The G. hirsutum GhGRFs gene family has 31 pairs of GRF homologues with A. thaliana, 14 pairs of homologues with O. sativa, 10 pairs of GRF homologues with T. aestivum, 34 pairs of GRF homologues with P. trichocarpa and nine pairs of GRF homologues with Z. mays.

2.4. GhGRFs Gene Family Cis-Element Analysis

To determine the expression pattern of the GhGRFs gene family, the cis-elements of the GhGRFs gene family promoters were analyzed using the PlantCARE database (Figure 5). These elements are involved in cotton growth and development as well as in stress response, including abiotic and biotic stresses, phytohormone responses, and plant growth and development. Stress-related cis-elements (MYB, MYC, ARE) were enriched in some GhGRF genes, suggesting that these GhGRF genes may play a key role in response to unfavorable conditions. In addition, the promoters of some GhGRF genes are enriched in ABRE (involved in ABA response), such as GhGRF29, which may be responsive to ABA hormones. The GhGRF10/13/29 promoter contains a large number of promoter elements related to growth and development, such as the G-box.

2.5. Transcriptome Analysis of GhGRF Gene Family in G. hirsutum

To further investigate the role of the GhGRFs gene family in cotton growth, development and abiotic stresses, we downloaded tissue expression and abiotic stress expression transcriptome data of the GRF gene family of G. hirsutum from the database. Expression patterns of the GhGRFs across 10 different tissues and under four abiotic stresses conditions were visualized using heatmaps (Figure 6). The results showed that most GhGRFs were downregulated under abiotic stress, including GhGRF8, GhGRF9, and GhGRF22. In contrast, GhGRF2 and GhGRF34 were upregulated under three of the stress treatments. Several genes exhibited tissue-specific high expression; for instance, GhGRF13 and GhGRF30 were highly expressed in leaves, while GhGRF18 showed elevated expression in filaments and petals. These findings suggest that members of the GhGRF gene family may perform distinct functions at different developmental stages and in response to environmental stresses in cotton.

2.6. GRF Gene Family Expression Patterns in Embryonic Callus (EC) and Non-Embryonic Callus (NEC)

Next, transcriptomic sequencing was performed on EC and NEC from G. hirsutum, which led to the identification of 12 GhGRFs genes. The results indicated that all of these genes were upregulated in EC compared to NEC (Figure 7A). Their expression patterns were further validated by RT-qPCR (Figure 7B,C). The results confirmed that GhGRF1, GhGRF13, GhGRF14, GhGRF16 and GhGRF20 were significantly highly expressed in EC but at lower levels in NEC, suggesting their potential key roles in EC formation and maintenance.
To further explore the involvement of GhGRFs in EC, we focused on seven genes that exhibited marked upregulation in the expression heatmap. A regulatory network analysis revealed that GhGRF1, GhGRF18, GhGRF13, GhGRF26, GhGRF14, GhGRF33, and GhGRF15 are predicted to be regulated by 8, 8, 12, 10, 10, 3, and 4 transcription factors, respectively (Figure 8A–G). These transcription factors belong to families such as MYB, Dof, ERF, HD-ZIP, AP2, etc. Additionally, the same gene may be co-regulated by the same transcription factor simultaneously. GhGRF1 and GhGRF18 potentially share eight common regulators. Since these transcription factors themselves show tissue-specific expression patterns, our findings imply that GhGRF genes may be differentially regulated across tissues. These transcription factors exhibit varying expression levels in different tissues, suggesting that GhGRF genes may be regulated by different transcription factors in different tissues.

3. Discussion

GRF genes constitute a plant-specific transcription factor family widely involved in growth development, and stress response, playing important regulatory roles in plants [22,36]. In this study, 34 GRF genes were identified in G. hirsutum. Notably, the number of GRF genes in G. hirsutum exceeds that reported in several model species, such as A. thaliana (9), P. trichocarpa (26), O. sativa (19), and T. aestivum (16) [24,26,29]. This variation suggests that the GRF family has undergone different expansion through duplication events during evolution. Analysis of the physicochemical properties of the GhGRFs indicated that most are basic hydrophilic proteins, with a Grand Average of Hydropathicity (GRAVY) below zero. Furthermore, an instability index greater than 40 for most members suggests these proteins may be relatively prone to degradation or denaturation (Table S1).
Phylogenetic analyses classified the 34 GhGRFs into four distinct clades, with members of the same subfamily showing highly similar homology (Figure 1A). There were significant differences in the number of GRF genes in different species, which reflected the diversity and adaptability of the GRF genes family during the evolutionary process (Figure 1B) [24]. Whole-genome duplication events have notably expanded the GRF family in some species [37]; for example, there are 48 GRF genes in tetraploid kale (Brassica napus), far more than its diploid ancestors [38], and Salix purpurea also exhibits a high GRF count likely due to genome duplication [29]. Furthermore, covariance analysis of gene families can explain gene evolution [39]. Tandem duplications and segmental duplications play a key role in species evolution [40]. In cotton, the GRF gene family forms two tandem duplication clusters on chromosomes A12 and D12 (Figure S1). Additionally, two segmental duplication pairs (GhGRF14/15 and GhGRF31/32) exhibited upregulation, supporting the role of both duplication types in the expansion of cotton GRF genes. Synteny analysis also revealed numerous orthologous gene pairs between cotton and five representative model plants, indicating a degree of evolutionary conservation across herbaceous and woody lineages (Figure 4).
Gene structure diversity often mirrors functional differentiation [41]. Exon–intron architecture analysis of GhGRFs revealed considerable variation in exon (1–23) and intron (0–23) numbers and arrangements, implying relatively low structural conservation and potential functional divergence among the 34 genes (Figure 2). This structural diversity is consistent with observations in other plants, highlighting the plasticity of the GRF family across species [42]. At the protein level, two conserved structural domains, QLQ and WRC, were predicted at the N-terminus of all GhGRFs, aligning with previous reports [43]. The QLQ domain typically mediates protein–protein interactions, including transcription factor complexes formation, whereas the WRC domain is involved in DNA binding [44]. It contributes to the recognition and binding of specific DNA sequences by transcription factors, thereby regulating gene expression. The conservation and functional relevance of these two structural domains underscore the likely importance of GhGRFs in regulating cotton growth, development and stress responses.
GRFs, as key regulators of plant growth, primarily by controlling cell proliferation and expansion during primary and secondary development [45]. In A. thaliana, for example, members of the GRFs form complexes with GIF (GRF-Interacting Factor) proteins to regulate cell division and organ formation [43]. Beyond development, GRFs also participate in hormone signaling and abiotic stress responses [42,46]. For example, miR396 regulates root growth and drought tolerance in plants under drought stress by targeting GRFs [22]. Gene expression levels are critical for plant growth, development, and response to external stresses [47]. Our expression analysis showed that GhGRF genes are broadly expressed across various tissues and developmental stages (Figure 6A), yet individual members exhibited distinct expression patterns in different organs and under abiotic stresses. This specificity may be linked to the varied cis-regulatory elements identified in their promoters (Figure 5 and Figure 6B). Notably, GhGRF promoters are enriched in stress-related elements, and their expression is highly responsive to abiotic stress. Furthermore, transcriptomic analysis of embryogenic callus (EC) and non-embryogenic callus (NEC) identified 12 GhGRFs that were consistently upregulated in EC (Figure 7). Their expression may be finely tuned by different transcription factors across tissues (Figure 8). Although the GRF gene family in upland cotton has been previously identified, we re-identified 34 GRF genes using the latest upland cotton genome data [42,48]. These results suggest that upregulation of specific GhGRFs, as key developmental regulators, may contribute to establishing and maintaining the high differentiation potential of embryogenic callus, which is essential for genetic transformation and plant regeneration in cotton.

4. Materials and Methods

4.1. Cotton Material Cultivation and Processing

The cotton material used in this experiment was sourced from the Cotton Research Institute of Hebei Academy of Agricultural and Forestry Sciences, variety JIN668 [6,49,50]. After removing the seed coats, the cotton seeds were disinfected in a 0.1% mercuric chloride solution for 8–10 min, then rinsed five times with ddH2O to thoroughly remove the surface mercuric chloride solution.
The seeds were then cultured in prepared MS medium (MS (Murashige and Skoog) 2.2 g/L, sucrose 20 g/L, pH adjusted to 7.8, agar 7 g/L) for 5–7 days after autoclaving (121 °C, 15 min). After 5–7 days, the cotton hypocotyls were cut into 0.5–1 cm segments and placed in callus induction medium (MS + 0.1 mg/L 2,4-Dichlorophenoxyacetic acid + 0.1 mg/L Kinetin + 3% glucose + 0.3% Phytagel, pH 5.85–5.95) for induction. Finally, culturing was continued until NEC and EC were obtained. All operations were performed in a sterile tissue culture workbench.

4.2. Identification of the GhGRF Genes Family in G. hirsutum and Construction of an Evolutionary Tree

The GhGRFs gene family was identified and analyzed based on the whole genome of G. hirsutum, HAU [51]. The G. hirsutum genome sequence was downloaded from the CottonFGD database (https://cottonfgd.net/). The GRF gene family files for species such as A. thaliana, T. aestivum, B. napus, G. soja, O. sativa (japonica), P. euphratica, P. trichocarpa, S. purpurea, and Z. mays are sourced from the Plant Transcriptional Regulation Atlas Database (https://plantregmap.gao-lab.org/) [52]. We searched for GhGRF genes from the terrestrial cotton genome using the Trp-Arg-Cys motif (WRC) and Gln, Leu, Gln motif (QLQ) structural domains and used the CottonFGD database to examine and obtain the correct GhGRF family genes [53,54]. The physicochemical properties of the GhGRF genes were obtained through the Expasy website (https://web.expasy.org/compute_pi/ (accessed on 4 August 2024)) [55].
For a more comprehensive analysis of the GhGRF genes family, a lineage tree was obtained by comparing all GhGRF protein sequences using ClustalW 2.1 [56,57]. The evolutionary tree was constructed using MEGA5.0 based on the 1000 bootstrap neighbor-joining (NJ) method [58]. Each GhGRF gene was named according to its position on the chromosome, and different subfamilies were indicated by different colors.

4.3. Analysis of Gene Structure, Conserved Motifs, and Cis-Acting Elements of the GhGRF Genes Family in G. hirsutum

The GhGRF genes family was analyzed bioinformatically as previously described. The GhGRF genes family was examined using The MEME Suite online website (https://meme-suite.org/meme/index.html (accessed on 15 September 2024)) to analyze its structure and identify conserved motifs [59]. Use the One Step Build a ML Tree feature in TBtools software (v2.423) to construct a phylogenetic tree, then obtain the Newick file [60,61]. Simultaneously, submit the amino acid sequences to NCBI-CDD for conserved domain analysis (https://www.ncbi.nlm.nih.gov/) [62,63].
In addition, GhGRFs were analyzed for cis-elements using the PlantCARE database, and graphical visualization was performed using Tbtools software [41].

4.4. Analysis of Gene Duplication and Expression Patterns of the GhGRFs Gene Family in G. hirsutum

Co-collinearity analyses between the G. hirsutum GhGRF gene family and G. hirsutum itself as well as other species were assessed using the Multiple collinearity Scan toolkit (MCScanX) [64]. In addition, abiotic stress transcriptome data and tissue expression transcriptome data were obtained from the CottonFGD database. Graphical visualization was performed using Tbtools software.

4.5. RT-qPCR Analysis

RNA extraction was performed using the Nuoto® AutoExtracter-32 Nucleic Acid Extractor with the 5fz PCR DNA/RNA AutoPurification Kit from Kangma-Healthcode (Shanghai, China). Extracted RNA was converted to cDNA using the Reverse Transcription Kit (Tiangen Biotechnology Co., Ltd., Beijing, China). Primers were designed by the Primer 5 software and synthesized by Sangon Ltd. in Shanghai, China. RT-qPCR experiments were performed with GhUBQ7 (Ghir_D12G021700) as internal reference genes (Table S3) [65]. Three biological replicates and four technical replicates were performed for each sample. RT-qPCR was performed on a CFX96 Touch™ instrument (Bio-Rad Co. Ltd., Hercules, CA, USA). RT-qPCR procedures and analysis of the results followed previous studies.

4.6. Bioinformatics Analysis and Tissue Expression Analysis of Upstream Transcription Factors of GhGRFs

Potential upstream transcription factors of GhGRFs were identified using the PlantRegMap database (http://plantregmap.gao-lab.org/network.php (accessed on 25 October 2024)) [66]. Network diagrams visualizing target genes and their upstream transcription factors were generated using Office software (2013). In the initial phase of the study, gene expression profiles of upstream transcription factors under different treatments were obtained from the CottonFGD database and visualized using TBtools software.

4.7. Statistical Analysis

We analyzed the experimental data using SPSS v.25.0 (SPSS Inc., Chicago, IL, USA). For gene relative expression, one-way analysis of variance (ANOVA) was performed (LSD multiple comparison test, * p < 0.05; ** p < 0.01). Prior to conducting the ANOVA, data were tested for normality and homogeneity of variance [47].

5. Conclusions

In this study, we performed a genome-wide identification of the GRF transcription factor family in G. hirsutum, characterizing 34 GhGRF genes. Comprehensive analyses revealed their gene structures, phylogenetic relationships, expression profiles across tissues, responses to abiotic stresses, and specific expression in embryogenic callus (EC). All identified GhGRF proteins contain the characteristic and highly conserved QLQ and WRC domains. Phylogenetic analysis classified the GhGRFs into four distinct clades (I–IV). These genes are distributed asymmetrically across 19 chromosomes, with expansion primarily driven by segmental duplication events, highlighting a key mechanism in the evolution of this family in cotton. Collinearity analysis indicated that the GhGRF family shares closer homology and a stronger evolutionary association with woody plants compared to herbaceous species. Cis-acting element analysis of the GhGRF promoters revealed a strong enrichment of stress-responsive elements (e.g., MYB, MYC, ARE, ABRE), supporting their potential involvement in abiotic stress signaling. Expression profiling demonstrated that GhGRF members exhibit distinct, often tissue-specific expression patterns and differential responses to various abiotic stresses, suggesting functional diversification and the regulation of separate signaling pathways. Transcriptomic sequencing of embryogenic (EC) and non-embryogenic callus (NEC) showed that 12 GhGRF genes were consistently upregulated in EC. RT-qPCR validation confirmed the significantly higher expression of GhGRF1, GhGRF13, GhGRF14, GhGRF16, and GhGRF20 in EC, pointing to their potential key roles in the establishment and maintenance of embryogenic competence. Furthermore, regulatory network analysis suggests that the expression of GhGRF genes is likely modulated by different sets of transcription factors in various tissues. In summary, our findings demonstrate that members of the GhGRF family are upregulated during somatic embryogenesis in cotton. The sustained and specific expression of different GhGRF transcription factors in embryogenic callus, potentially through distinct regulatory pathways, confers diverse functions. This underscores their indispensable role within the complex regulatory network governing callus formation and somatic embryogenesis in G. hirsutum.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27052191/s1.

Author Contributions

C.-H.F., J.C., D.L., J.Z., M.L., M.J., and L.L. designed the experiments. C.-H.F., J.C., and L.L. drafted the manuscript. C.-H.F. and L.L. conducted RT-qPCR and data analysis. J.C., D.L., L.L., M.L., J.Z. and C.-H.F. critically reviewed the manuscript, L.L. and J.Z. contributed to project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Biological Breeding of Stress tolerant and High Yield Cotton Varieties (Project NO.2023ZD04040-2), and HAAFS Agriculture Science and Technology Innovation Project (2022KJCXZX-MHS-7, 2026KJCXZX-MHS-4).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. Phylogenetic analysis of the GRF gene family in G. hirsutum and the number of GRF genes across multiple species. (A) Evolutionary and phylogenetic analysis of the GhGRFs gene family in G. hirsutum. The GRF gene family in G. hirsutum is divided into four subfamilies based on different colors. (B) Number of GRF gene family members across 14 species. Arabidopsis thaliana; Triticum aestivum; Brassica napus; Glycine soja; Gossypium hirsutum; Oryza sativa (japonica); Populus euphratica; Populus trichocarpa; Salix purpurea; Zea mays; Theobroma cacao; Glycine max; Coffea canephora; Marchantia polymorpha.
Figure 1. Phylogenetic analysis of the GRF gene family in G. hirsutum and the number of GRF genes across multiple species. (A) Evolutionary and phylogenetic analysis of the GhGRFs gene family in G. hirsutum. The GRF gene family in G. hirsutum is divided into four subfamilies based on different colors. (B) Number of GRF gene family members across 14 species. Arabidopsis thaliana; Triticum aestivum; Brassica napus; Glycine soja; Gossypium hirsutum; Oryza sativa (japonica); Populus euphratica; Populus trichocarpa; Salix purpurea; Zea mays; Theobroma cacao; Glycine max; Coffea canephora; Marchantia polymorpha.
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Figure 2. Analysis of motifs, conserved domains, and gene structures in the GhGRFs gene family of G. hirsutum. (A) Phylogenetic tree of the GhGRFs gene family in G. hirsutum; (B) Motif analysis of the GhGRFs gene family in G. hirsutum, with each motif represented by a differently colored square; (C) Gene structure analysis of the GhGRFs gene family in G. hirsutum; (D) Gene structure analysis of the GhGRFs gene family in G. hirsutum, with upstream and downstream non-coding regions indicated by green rectangles, exons by yellow rectangles, and introns by gray lines.
Figure 2. Analysis of motifs, conserved domains, and gene structures in the GhGRFs gene family of G. hirsutum. (A) Phylogenetic tree of the GhGRFs gene family in G. hirsutum; (B) Motif analysis of the GhGRFs gene family in G. hirsutum, with each motif represented by a differently colored square; (C) Gene structure analysis of the GhGRFs gene family in G. hirsutum; (D) Gene structure analysis of the GhGRFs gene family in G. hirsutum, with upstream and downstream non-coding regions indicated by green rectangles, exons by yellow rectangles, and introns by gray lines.
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Figure 3. Collinearity analysis of the GhGRFs gene family. The fan-shaped rings indicate chromosome numbers and sizes. Gray line segments represent genomic collinear blocks, while red lines denote homologous gene pairs of GhGRF genes.
Figure 3. Collinearity analysis of the GhGRFs gene family. The fan-shaped rings indicate chromosome numbers and sizes. Gray line segments represent genomic collinear blocks, while red lines denote homologous gene pairs of GhGRF genes.
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Figure 4. Collinearity analysis of the GhGRFs gene family in G. hirsutum and 5 plant species. (A): A. thaliana; (B): O. sativa; (C): T. aestivum; (D): P. trichocarpa; (E): Zea mays. Gray lines indicate the collinearity between the G. hirsutum genome and those of A. thaliana, O. sativa, T. aestivum, P. trichocarpa, and Z. mays, while red lines highlight collinear gene pairs between G. hirsutum GhGRF genes and genes from other species.
Figure 4. Collinearity analysis of the GhGRFs gene family in G. hirsutum and 5 plant species. (A): A. thaliana; (B): O. sativa; (C): T. aestivum; (D): P. trichocarpa; (E): Zea mays. Gray lines indicate the collinearity between the G. hirsutum genome and those of A. thaliana, O. sativa, T. aestivum, P. trichocarpa, and Z. mays, while red lines highlight collinear gene pairs between G. hirsutum GhGRF genes and genes from other species.
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Figure 5. Promoter cis-acting elements of GhGRFs genes. (A) The heatmap represents the number of cis-acting elements; (B) The histogram shows the number of cis-acting elements of different types per gene, with different colors denoting distinct categories: plant hormone response type, abiotic and biotic stress type, and plant growth and development type.
Figure 5. Promoter cis-acting elements of GhGRFs genes. (A) The heatmap represents the number of cis-acting elements; (B) The histogram shows the number of cis-acting elements of different types per gene, with different colors denoting distinct categories: plant hormone response type, abiotic and biotic stress type, and plant growth and development type.
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Figure 6. Expression profiles of GhGRFs during G. hirsutum growth and development and under abiotic stresses. (A) Heatmap of GhGRFs gene expression levels in different tissues. (B) Heatmap of GhGRFs gene expression levels under cold stress, drought stress, heat stress, and salt stress. The color bars represent high and low relative expression levels, respectively.
Figure 6. Expression profiles of GhGRFs during G. hirsutum growth and development and under abiotic stresses. (A) Heatmap of GhGRFs gene expression levels in different tissues. (B) Heatmap of GhGRFs gene expression levels under cold stress, drought stress, heat stress, and salt stress. The color bars represent high and low relative expression levels, respectively.
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Figure 7. Transcriptome and RT-qPCR analysis of GhGRFs gene expression in EC and NEC. (A) RNA-seq analysis of 12 GhGRF genes in EC and NEC. The color bars represent high and low relative expression levels, respectively. (B,C) RT-qPCR results for 12 GhGRF genes in G. hirsutum EC and NEC. One-way ANOVA with LSD multiple comparison, n = 12. * p < 0.05; ** p < 0.01.
Figure 7. Transcriptome and RT-qPCR analysis of GhGRFs gene expression in EC and NEC. (A) RNA-seq analysis of 12 GhGRF genes in EC and NEC. The color bars represent high and low relative expression levels, respectively. (B,C) RT-qPCR results for 12 GhGRF genes in G. hirsutum EC and NEC. One-way ANOVA with LSD multiple comparison, n = 12. * p < 0.05; ** p < 0.01.
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Figure 8. Bioinformatics analysis of upstream transcription factors (TFs) of GhGRFs. (A–G) Mutual regulation network analysis of TFs-GhGRF1/18/13/26/14/33/15. Green circles indicate upstream transcription factors of GhGRFs; yellow circles represent GhGRF1/18/13/26/14/33/15. Heatmap showing transcriptional levels of upstream transcription factors in leave, root, and stem. The color bars represent high and low relative expression levels, respectively.
Figure 8. Bioinformatics analysis of upstream transcription factors (TFs) of GhGRFs. (A–G) Mutual regulation network analysis of TFs-GhGRF1/18/13/26/14/33/15. Green circles indicate upstream transcription factors of GhGRFs; yellow circles represent GhGRF1/18/13/26/14/33/15. Heatmap showing transcriptional levels of upstream transcription factors in leave, root, and stem. The color bars represent high and low relative expression levels, respectively.
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Feng, C.-H.; Liu, L.; Liu, D.; Zhen, J.; Li, M.; Jiang, M.; Chi, J. Genome-Wide Identification and Expression Analysis of the GRF Gene Family in Gossypium hirsutum L. Int. J. Mol. Sci. 2026, 27, 2191. https://doi.org/10.3390/ijms27052191

AMA Style

Feng C-H, Liu L, Liu D, Zhen J, Li M, Jiang M, Chi J. Genome-Wide Identification and Expression Analysis of the GRF Gene Family in Gossypium hirsutum L. International Journal of Molecular Sciences. 2026; 27(5):2191. https://doi.org/10.3390/ijms27052191

Chicago/Turabian Style

Feng, Cong-Hua, Linlin Liu, Di Liu, Junbo Zhen, Mengzhe Li, Mengmeng Jiang, and Jina Chi. 2026. "Genome-Wide Identification and Expression Analysis of the GRF Gene Family in Gossypium hirsutum L." International Journal of Molecular Sciences 27, no. 5: 2191. https://doi.org/10.3390/ijms27052191

APA Style

Feng, C.-H., Liu, L., Liu, D., Zhen, J., Li, M., Jiang, M., & Chi, J. (2026). Genome-Wide Identification and Expression Analysis of the GRF Gene Family in Gossypium hirsutum L. International Journal of Molecular Sciences, 27(5), 2191. https://doi.org/10.3390/ijms27052191

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