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Article

Genome-Wide Identification of the Growth-Regulating Factor (GRF) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii

by
Yan Deng
1,
Yun Pan
1,
Fei Wang
1,
Feng Chen
1,
Xiaopei Wu
1,
Jinliao Chen
1,
Jin Zhu
1,2,* and
Donghui Peng
1,*
1
Key Laboratory of National Forestry and Grassland Administration for Orchid Conservation and Utilization, College of Landscape Architecture and Art, The Innovation and Application Engineering Technology Research Center of Ornamental Plant Germplasm Resources in Fujian Province, National Long Term Scientific Research Base for Fujian Orchid Conservation, Fujian Agriculture and Forestry University, Fuzhou 350002, China
2
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1015; https://doi.org/10.3390/horticulturae11091015
Submission received: 6 July 2025 / Revised: 22 August 2025 / Accepted: 25 August 2025 / Published: 27 August 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Browse Figures
Versions Notes

Abstract

The GRF (Growth-Regulating Factor) gene family has indispensable regulatory functions in the morphological and physiological development of plants. Nonetheless, comprehensive investigations of GRF gene family members and their functional roles in Cymbidium goeringii, Cymbidium ensifolium, and Cymbidium sinense are still lacking. Therefore, the GRF gene family members in three Cymbidium species were systematically identified, and their expression profiles and potential biological functions were comprehensively evaluated in the study. The results provided evidence that eleven, eleven, and nine GRF genes were identified in C. goeringii, C. ensifolium, and C. sinense, respectively. These genes encode proteins considered as 153–584 amino acids and have been postulated to be located in the cell nucleus. The promoter contains cis-acting elements associated with hormone response regulation, tissue-specific expression, modulation of organismal growth and development, and environmental signal response. The analyses of gene architecture and motif composition demonstrated that introns and motifs within each evolutionary branch are highly similar, whereas significant differences exist between evolutionary branches. The results of chromosome localization and collinearity analysis showed that only a pair of segmental duplication genes was identified in C. goeringii. Moreover, transcriptome data and qRT-PCR results indicated that GRF genes are involved in various organs of C. goeringii. In conclusion, these findings may establish a foundation for theoretical inquiry into the future functional analysis of GRF genes in orchids.

1. Introduction

Growth-regulating factors (GRFs) are a class of a plant-specific family of transcription factors implicated in diverse developmental processes, characterized by two highly conserved domains, QLQ and WRC, located in the N-terminal region of their protein-coding sequences. These factors are essential for various developmental processes, including roots, stems, leaves, and flower formation [1]. Additionally, GRFs are involved with the regulation of morphological and developmental traits, particularly under stressful environmental conditions [2]. The function of Oryza sativa L. and Triticum aestivum GRF has been studied well [3,4,5,6]. The first plant GRF gene, OsGRF1, was identified in Oryza sativa in 2000 [7]. At present, the functions of GRF genes in multiple plant species have been widely studied, especially in woody plants [8], tuberous plants [9], and fruit trees [10,11]. We have a relatively sound understanding of the role of GRF genes in plant growth and differentiation, especially in regulating plant morphology, development, and fruit cell expansion. For example, the PagGRF11 gene in Populus is extensively involved in growth processes, with its overexpression resulting in reduced plant height, increased stem diameter, shortened internodes, and enlarged leaf area [8]. The StGRF gene family exhibits high expression in roots, shoot tips, flowers, and young tubers. Among them, seven GRF genes contribute to tuber sprouting, with StGRF4 and StGRF9 playing important roles in regulating the sprouting process [9]. In Citrus, CsGRF1, CsGRF4, CsGRF5, CsGRF6, and CsGRF8 regulate fruit cell expansion during development, with CsGRF3 and CsGRF4 being essential for rapid cell expansion in fruit [11]. However, research on GRF genes in ornamental plants remains limited, and related studies are still in their preliminary stage.
Orchids represent one of the most valuable ornamental crops in the global horticultural market, prized for their diverse floral forms, vivid colors, and prolonged blooming periods [12]. However, the long vegetative growth cycle and slow breeding process significantly limit the rapid development of the orchid industry. Molecular breeding may provide an effective approach to accelerating genetic improvement and promoting cultivar innovation of orchids. Growth-Regulating Factors (GRFs) are known to play essential roles in regulating cell division [13], organ growth [14], and floral differentiation [15] in many plant species [16]. Previous studies have emphasized the major functional significance of GRF genes in developmental and growth-related pathways of orchids, particularly in regulating processes such as cell division, proliferation, and expansion [17]. Furthermore, GRFs play a critical role in the initial phases of floral organogenesis, regulating the differentiation of floral buds, and influencing the floral organ formation in C. ensifolium, which ensure the proper spatiotemporal development of floral organs by activating or inhibiting the expression of related genes [18]. Therefore, in-depth research on the GRF gene family could enhance the ornamental value of orchids. The genus Cymbidium exhibits distinct phenotypic characteristics, providing a valuable model system for exploring morphological diversity within orchids [19]. A comprehensive study of GRFs in Cymbidium could provide novel insights into the genetic regulation of growth development and offer potential targets for improving the ornamental qualities of orchid cultivars. To date, limited information is available regarding the evolutionary and functional characterization of the GRF gene family in Cymbidium.
In this study, the GRF genes were identified from the whole-genome sequences of three Cymbidium species (C. goeringii, C. ensifolium, and C. sinense). An in-depth examination of these GRF genes was conducted, including sequence structure, chromosomal localization, phylogenetic relationships, synteny analysis, and cis-regulatory element analysis. Additionally, the expression patterns of GRF genes in different tissues of C. goeringii were also investigated. This study aims to establish a foundation for further investigation into the role of GRF genes in the development and differentiation of organs in Cymbidium species.

2. Materials and Methods

2.1. Identification of the GRF Gene Family in Three Cymbidium Species

The full sequence of the proteins of C. goeringii, C. ensifolium, and C.sinense were obtained from the whole-genome sequencing information of the three species of Cymbidiums [20,21]. The protein sequences of GRFs were obtained from the genomic data of Arabidopsis thaliana, Oryza sativa, and Phalaenopsis equestris, which were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 27 March 2025). As the query sequence, the protein sequences of the GRF gene of A. thaliana were applied, and the GRF protein sequences of A. thaliana were in turn blasted in the three Cymbidium species with the TBtools software (V2.0). The prediction of whether the typical QLQ/WRC domains were present in the candidate genes was obtained by further analysis with Conserved Domain Database (CDD) search tool via NCBI (https://www.ncbi.nlm.nih.gov/cdd, accessed on 27 March 2025). Proteins that have inaccurate and insufficient QLQ/WRC segments were removed. Upon completion of the genome-wide screening, putative GRF gene family members were systematically identified across the three Cymbidium species.

2.2. Physicochemical Property Analysis and Phylogenetic Analysis

The protein physicochemical parameters of the GRF genes of the three Cymbidium species were predicted using the ExPasy website (https://web.expasy.org/compute_pi/, accessed on 2 April 2025), and the subcellular localization of these genes was predicted using the Plant-mPLoc online program. The GRF protein sequences from six species (A. thaliana, O. sativa, P. equestris, C. goeringii, C. ensifolium, and C. sinense) were aligned using MEGA software (V12.0) to facilitate comparative sequence analysis, based on previously identified sequences. Maximum likelihood (ML) was used for phylogenetic analysis, the Jones–Taylor–Thornton (JTT) model was selected for sequence permutation, and 1000 bootstraps were performed to evaluate the reliability of the phylogenetic tree. FigTree v1.4.3 was utilized to enhance the visual representation of the final phylogenetic tree.

2.3. Gene Structure Analysis and Conserved Domain of GRF

The gene structure was analyzed using TBtools. The exon–intron structure of the GRF gene was identified and mapped by aligning the cDNA and genomic sequences of the GRF gene. The protein sequences of CgGRF, CeGRF, and CsGRF were aligned and analyzed using the ClustalW function of Mega12, and the residues were colored using Jalview. The MEME online program (https://meme-suite.org/meme/index.html, accessed on 8 April 2025) identified the conserved motifs that are affiliated with the typical QLQ/WRC domain and visualized the conserved motifs corresponding to the GRF proteins of the three Cymbidium species.

2.4. Chromosome Localization and Colinearity Analysis

Tbtools software (V2.0) was used to locate and visualize the chromosomes of GRF genes in three Cymbidium species. Based on the results of gene alignment, segmental duplication genes were detected and visualized using Advanced Circos to display their linkage relationships and illustrate the intraspecific collinearity within the GRF gene family [22].

2.5. Cis-Acting Element Prediction

Cis-acting regulatory elements within the promoter regions of GRF genes were predicted using the PlantCARE database, and the results were subsequently visualized with Microsoft Excel 2010.

2.6. qRT-PCR Analysis

C. goeringii samples were collected from individuals cultivated at Fujian Agriculture and Forestry University, including roots, rhizomes, leaves, petals, sepals, lips, and gynostemiums in full bloom. Total RNA from the Cymbidium goeringii was extracted using the FastPure® Plant Total RNA Extraction Kit (for samples rich in polysaccharides and polyphenols) (Vazyme, Nanjing, China). The RNA quality was quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Shanghai, China), and good quality was determined in agarose gel electrophoresis. cDNA corresponding to the first strand was synthesized by reverse transcription of RNA samples using the HRbio™ III 1st Strand cDNA Synthesis SuperMix (HeRui, Nanjing, China) for qRT-PCR, which includes the OneStep gDNA Removal kit to eliminate genomic DNA during the process. Oligo 7.0 was used to design primers, and the reference gene was CgActin [23,24]. The detailed information on thereference gene and the primers is listed in Table S1. HRbio™ qRT-PCR SYBR Green Master Mix (Low Rox Plus) (HeRui, Nanjing, China) was used for qRT-PCR reactions. Relative gene expression was determined by the 2−∆∆Ct method [25]. A one-way analysis of variance (ANOVA) was performed on the IBM SPSS STATISTICS version 27.0 to test the statistical difference between all the extracted different samples of tissues. Duncan’s multiple range test was then used to determine which groups exhibited significant differences in mean values. The significance levels were given by p < 0.05.

3. Results

3.1. The Characterization of the GRF Genes in Three Cymbidium Species

An overall number of 31 GRF genes were identified across three Cymbidium species, with 11 genes in C. goeringii, 11 in C. ensifolium, and 9 in C. sinense (Table 1). The genes were labeled based on their chromosomal positions as CgGRF1 to CgGRF11 for C. goeringii, CeGRF1 to CeGRF11 for C. ensifolium, and CsGRF1 to CsGRF5 and CsGRF8 to CsGRF11 for C. sinense. Upon analyzing the properties of these genes, it was found that their protein sequences range in length from 153 to 585 amino acids. The calculated isoelectric points (PI) of the proteins span from 7.18 to 10.06, and their instability indices (II) lie between 50.25 and 75.47. The molecular weights (Mw) for these proteins are between 16.70 kD and 65.11 kD, while their aliphatic indices (AI) range from 41.06 to 75.47. The Grand Average of Hydropathicity [26] (GRAVY) for the proteins vary from −1.057 to −0.433, suggesting that they are predominantly hydrophilic. The result of subcellular localization prediction showed that all GRFs of three Cymbidium species might exist in the cell nucleus.

3.2. Phylogeny Analysis of the GRF Genes

The evolutionary relationship of the GRF gene family was established through a comparison of the protein sequences from A. thaliana, O. sativa, P. equestris, C. goeringii, C. ensifolium, and C. sinense (Figure 1). The 60 GRF genes from these six selected species were classified into six major branches, designated Clade A–F. Clade A consists of two AtGRFs, three OsGRFs, four PeGRFs, four CsGRFs, four CgGRFs, and three CeGRFs. Clade B includes only dicots and orchids. Clade C consists solely of monocots, containing three OsGRFs, three CsGRFs, three CgGRFs, two CeGRFs, and two PeGRFs. Clade D contained two AtGRFs, three OsGRFs, three CgGRFs, four CeGRFs, three CsGRFs, and two PeGRFs. Clade E comprises a single CgGRF. Clade F consisted of one AtGRF, two OsGRFs, one CeGRF, and one CgGRF. These results highlight the uneven distribution of GRF proteins across the six clades for the six species.

3.3. Conserved Domain and Gene Structure Analysis of GRF

The multiple sequence alignments of the GRF domains of C. goeringii, C. ensifolium, and C.sinense were performed, and the result indicated that the GRF genes of the three species of Cymbidium possessed two absolutely conserved domains, WRC and QLQ (Figure 2). With the MEME online tool, it was found that GRF gene family had 10 preserved motifs, and the presence of conserved motifs and their consistent arrangement within the genes demonstrates the similarity in gene structure across the GRF genes and GRF genes having the same conserved motifs showed a tendency to gather within the same evolutionary branch in the phylogenetic tree (Figure 3A). The results reflected that both typical motifs, motif 1 (WRC) and motif 2 (QLQ), were present in all the GRF group of genes, meaning the two motifs are still relatively conserved in the GRF gene family. It was found that in the 31 GRF genes tested, 10 different conserved motifs were predicted. Motif 1 and 2 showed common conserved regions in all the GRFs. It is worth noting that the CeGRF4, CsGRF4, CgGRF4, and CgGRF11 genes each contained the most of these conserved motifs, with nine each (Figure 3B). This is an indication of the fact that these genes are grouped according to structural and motif characteristics, which can be said to be preserved by conserved functions or controlling processes in the three Cymbidium species.

3.4. Cis-Acting Element Analysis of Three Cymbidium Species

To further investigate the regulatory mechanisms of GRF genes in three species of Cymbidium, and to identify potential cis-regulatory elements, the promoter regions 2000 bp upstream of the GRF genes were predicted. In the 2000 bp promoter sequences of the 11 GRF genes of C. goeringii, 250 cis-acting elements were identified, with their distribution and quantity displayed in Figure 4A. In the 2000 bp promoter sequences of 11 GRF genes of C. ensifolium, 247 cis-acting elements were identified, with their distribution and quantity shown in Figure 4B. In the 2000 bp promoter sequences of nine GRF genes of C. sinense, 185 cis-acting elements were identified, with their distribution and quantity shown in Figure 4C. In the GRF gene families of Cymbidium goeringii, Cymbidium ensifolium, and Cymbidium sinense, light responsiveness (50.0%, 48.9%, 50.8%), MeJA responsiveness (11.9%, 9.3%, 9.7%), anaerobic induction (7.9%, 6.2%, 7.6%), and MYB binding site (4%, 4.1%, 3.8%) elements were identified (Figure S3). Of these, light response elements were found to be the most abundant, followed by MeJA responsiveness and anaerobic induction. This suggests that light response elements interact with plant hormone signaling pathways, regulating the synthesis and transduction of plant hormones under light conditions. A substantial quantity of responsive elements and cis-acting elements related to stress were found in the promoter domains of the GRF family in all three Cymbidium species. The main elements included auxin responsiveness (5, 3, 2), defense, and stress responsiveness elements (12, 6, 7), gibberellin responsiveness (7, 11, 7), and low-temperature responsiveness elements (7, 8, 4). It can be inferred from the results that GRFs may be crucial for the growth and development of the three Cymbidium species studied, as well as for other species within Cymbidium.

3.5. Chromosome Location and Collinearity Analysis

This study evaluated the chromosomal distribution of GRF genes in three species of Cymbidium by constructing gene distribution maps. The distribution of GRF on chromosomes of the three Cymbidium species is shown in Figure S2. In C. goeringii, the CeGRF3 and CeGRF6 genes were co-localized on chromosome Chr1, while the other GRF genes were mapped to distinct chromosomes.
Additionally, a collinearity analysis of the GRF gene sequences across the three Cymbidium species was conducted. The results revealed that the C. ensifolium and C. sinense genomes lacked any segmental duplicated genes (Figure 5B,C). In contrast, C. goeringii showed corresponding one-to-one relationships for the GRF genes found in the other two Cymbidium species. A pair of segmental duplicated genes was found in the C. goeringii genome, located on Chr08 and Chr20, identified as CgGRF11 and CgGRF4 (Figure 5A).

3.6. Expression Pattern of GRF Genes in Three Cymbidium Species

Expression profiles of 11 GRF genes across various tissues of C. goeringii were examined using FPKM-based transcriptomic data, including roots, stems, leaves, sepals, petals, lips, and gynostemium. The results indicated that the expression levels of CgGRF genes varied across the eight different samples (Figure 6A, Table S4). CgGRF5, CgGRF6, CgGRF7, CgGRF8, and CgGRF9 exhibited higher expression levels in roots compared to the other five tissues. GRF2, GRF4, and GRF11 showed higher expression in the gynostemium, with moderate or low expression in other tissues. GRF1 exhibited moderate expression in the lip and low expression in the gynostemium. The analysis revealed that CgGRF3, CgGRF4, CgGRF5, CgGRF9, and CgGRF10 may play a critical role in root development of C. goeringii.
In order to study the expression pattern of GRF genes in various tissues of C. goeringii, six genes were selected based on the significant differences in the FPKM values of these genes in different tissues’ CgGRF1, CgGRF2, CgGRF4, CgGRF5, CgGRF7, and CgGRF8 genes for qRT-PCR analysis. Evidence from the data suggested that these six genes were all expressed in roots, leaves, sepals, petals, lip, and gynostemium (Figure 6B), indicating that they were involved in the development of different organs in C. goeringii. The expression levels of CgGRF2, CgGRF5, CgGRF7, and CgGRF8 in C. goeringii were the highest in the roots, followed by lip, leaf, petal, and sepal. CgGRF1 exhibited its peak expression in the lip, with relatively elevated transcript levels also detected in the roots, leaves, and petals, while its expression was minimal in the sepals.

4. Discussion

GRF transcription factors are key regulators of plant growth. A total of 31 GRF genes were identified in this study from three Cymbidium species, including 11 CgGRFs from C. goeringii, 11 CeGRFs from C. ensifolium, and 9 CsGRFs from C. sinense (Table 1). Phylogenetic analysis revealed that the 60 GRF genes could be classified into six branches. However, Arabidopsis thaliana exhibited four branches, with the loss of Clades C and E; Oryza sativa was divided into four branches, but no genes were located in Clades B and E. Phalaenopsis equestris only had three branches: Clades A, C, and D. Notably, Clade A contained the highest number of members among the six branches, with 19 GRF members, including 4 CgGRFs from C. goeringii, 3 CeGRFs from C. ensifolium, 3 CsGRFs from C. sinense, 2 AtGRFs from Arabidopsis thaliana, 4 OsGRFs from Oryza sativa, and 3 PeGRFs from P. equestris. The gene loss of specific species within a clade may be attributed to differences in gene contraction and evolutionary patterns among different plant species. It may also result from the mutual exclusion of certain genes, exhibiting PVA (presence/absence) characteristics [27]. The genes of three Cymbidium species within the same branch (Figure 1) exhibit identical gene structures and motifs (Figure 3). Previous studies have highlighted the functional roles of GRF genes in Arabidopsis thaliana and Oryza sativa, with genes displaying similar functions located within the same evolutionary branch [28,29,30], suggesting that GRF genes in the same branch may have similar functions.
The identification of this gene as a GRF gene was based on the presence of two key structural domains: the WRC and QLQ domains. Analysis of the GRF gene structures of the three Cymbidium species revealed that the number of exons ranged from 3 to 5, and the number of introns ranged from 2 to 4, with little difference in their distribution. This suggests that the GRF gene family among the three Cymbidium species is highly conserved during evolution. No segmental duplication genes of the GRF family were found in C. ensifolium and C. sinense. A pair of colinear genes was found in C. goeringii, which indicates that there may be a high degree of evolutionary conservation in C. goeringii, and their functions may be retained during species differentiation. Previous studies have shown the function of OsGRF4 and OsGRF11 in controlling grain shape and panicle length in Oryza sativa [31]. In this study, GRF genes were named based on their functions. The reference species for CgGRF4 and CgGRF11 were OsGRF4 and GRF11, respectively. It can be speculated that the collinearity between the two genes in C. goeringii may be due to similar functions.
Earlier research has demonstrated that GRF genes play a key role in the transduction of hormone signals. For example, the GRF gene in Phyllostachys edulis participates in hormone responses during development, with PheGRF4 initiating auxin signaling by binding to PheIAA30, further expanding the potential regulatory functions of the PheGRF genes [16]. In the present study, several cis elements related to hormone signal transduction, such as auxin responsiveness, gibberellin responsiveness, and salicylic acid response elements, were identified in the GRFs of the three Cymbidium species. The evidence points to the possibility that the GRF gene family in the three Cymbidium species is integral to stress response [32]. Moreover, the GRF expression could be adjusted in response to environmental cues. The expression levels of CrGRF1 and CrGRF4 are significantly higher under full sunlight than under shade in Catharanthus roseus [33]. The proportion of light response elements in the GRF genes of three Cymbidium species was the largest in the study, indicating that light response elements are directly involved in the response of GRF genes to light signals and may play a key role in regulating the adaptation of Cymbidium species to changes in environmental light conditions. Similarly, stress-responsive elements in the GRF gene promoter cause upregulation of GRF gene expression in Melastoma serrata under stresses such as drought and low temperature [34]. In this study, some GRF gene promoters of three species of Cymbidium also contained stress response elements, such as low-temperature responsiveness elements, salicylic acid responsiveness, and drought stress, indicating that these GRF genes may adapt to adverse environments by rapidly adjusting gene expression and coordinating physiological and biochemical processes when Cymbidium are subjected to abiotic stress. Other GRF genes also play critical roles in organ development, regulating leaf development, aging, inflorescence and root development, grain size, and the regeneration of plants [11,35]. Certain development-related cis elements, such as meristem expression, seed-specific regulation, and endosperm expression, have been identified in the GRFs of the three Cymbidium species. Previous research has shown that GRF genes exhibit varying expression levels across different tissues, including the root, stem, flower buds, and fruit [6]. For example, GRFs promote sugar accumulation in pear fruit by phosphorylating PbCPK28 and enhancing its activity, thus regulating plant growth and development [36]. In C. ensifolium, of the eleven CeGRF genes, ten are highly expressed in rapidly developing flower buds, underscoring the critical role of GRFs in floral development, particularly in the formation and growth of the gynostemium structure [37].
The expression patterns of specific genes across various tissues provide valuable information about their potential functions within those tissues. This study specifically investigated the expression of 11 selected CgGRF genes in C. goeringii, with a particular emphasis on the roots. (Figure 6A). The results showed that CgGRF1, CgGRF2, CgGRF4, CgGRF5, CgGRF7, and CgGRF8 exhibit high expression levels in multiple tissues. In the analysis of conserved motifs in GRF genes of three Cymbidium species, CgGRF4 and CgGRF11 were found to contain motif 9, whereas other GRF genes in C. goeringii did not exhibit it. Notably, the expression levels of CgGRF4 and CgGRF11 were significantly higher in the gynostemium tissues, suggesting that motif 9 may be closely associated with gene expression in gynostemium tissues. Furthermore, the presence and combination of different motifs may have varying effects on gene expression levels across different tissues. The cis elements have shown that CgGRF4 and CgGRF11 from the same evolutionary branch have the same type of cis elements, including MEJA responsiveness, defense and stress responsiveness, auxin responsiveness, gibberellin responsiveness, and low temperature. The result may contribute to their expression being higher in the gynostemium tissue. In the evolutionary analysis, CgGRF1 and CgGRF8 clustered with OsGRF1, while OsGRF10 and CgGRF4 formed a distinct cluster. Previous studies have demonstrated that the high expression of GRF genes in roots is particularly prominent in Arabidopsis, where AtGRF1 and AtGRF2 also control cotyledon development and leaf formation [18]. In Cymbidium ensifolium, transcriptomic data reveal that most CeGRF genes are predominantly expressed in flowers and gynostemium tissues [37]. In O. sativa, OsGRF1 is primarily expressed in young leaves containing leaf primordia, and its expression level can influence both internal and external conditions for leaf growth [29]. Moreover, OsGRF6 and OsGRF10 have been implicated in the regulatory pathways governing floral organ development [38]. These expression patterns and evolutionary similarities suggest that these genes may play important roles in Cymbidium and warrant further investigation. qRT-PCR analysis confirmed that CgGRF1, CgGRF5, and CgGRF8 exhibited their peak expression in the root, leaf, and column tissues (Figure 6B). Previous studies across various plant species have established that GRF genes are involved in key developmental processes, including floral organ development and meristem maintenance in both dicots and monocots [39,40]. Taken together, these observations imply that GRF genes in the three Cymbidium species may have conserved functions in modulating floral organogenesis. The collaboration between GRF genes and GIF plays a pivotal role in the development of maize and foxtail millet [41]. The integration of GRF transcription factors with other transcription factors (TFs) and cytokinin signaling [42,43] could promote plant development. Regulating the expression of GRF genes could alter the morphology of ornamental plants [2,25,44], thereby enhancing their ornamental and market value, ultimately advancing the progress of flower breeding.

5. Conclusions

This study preliminarily identified 11 GRF genes, 11 GRF genes and 9 GRF genes, respectively, in C. goeringii, C. ensifolium, and C. sinense. A comprehensive analysis was performed on the physicochemical properties, phylogeny, conserved motifs, gene structure, cis-regulatory elements, chromosomal localization, and collinearity of these 31 GRF genes. Transcriptome profiling suggests that CgGRF2, CgGRF5, CgGRF7, and CgGRF8 may exert regulatory effects on the root growth of C. goeringii while CgGRF2 and CgGRF4 may promote the development of the gynostemiums of C. goeringii. The results of qRT-PCR also support this point. These findings contribute to understanding the functional mechanisms of the Cymbidium GRF genes and provide a potential foundation for functional identification, as well as the future research directions in the breeding of new orchid varieties with higher ornamental value.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11091015/s1. Text S1: GRF protein sequences of A. thaliana, Oryza sativa, P. equestris, C. goeringii, C. ensifolium, and C. sinense; Table S1: The primers for qRT-PCRs; Table S2: GRF genes used for the phylogenetic tree in A. thaliana, Oryza sativa, P. equestris, C. goeringii, C. ensifolium, and C. sinense; Table S3: The Seqlogo of motifs; Table S4: The FPKM values of the CgGRF genes in different tissues; Figure S1: Analysis of GRF gene structure in three Cymbidium species; Figure S2: Distribution of CgGRFs, CeGRFs, and CsGRFs on chromosomes; Figure S3: The number and distribution of cis-element types in (A) C. goeringii, (B) C. ensifolium, and (C) C. sinense; Figure S4: The exons and introns of GRF genes in three Cymbidium Species.

Author Contributions

Conceptualization, Y.D., J.C. and D.P.; Data curation, F.C. and X.W.; Formal analysis, Y.P. and F.W.; Funding acquisition, D.P.; Resources, F.C. and X.W.; Supervision, J.C., J.Z. and D.P.; Writing—original draft, Y.D.; Writing—review and editing, Y.D., Y.P. and F.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Program of China (grant No. 2023YFD1600504).

Data Availability Statement

Data are contained within the article and supplementary materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Horticulturae 11 01015 g001
Figure 1. Phylogenetic tree of the GRF genes of six species. Protein sequences from A. thaliana, P. equestris, O. sativa, C. goeringii, C. ensifolium, and C. Sinensis. The phylogenetic tree was constructed using MEGA 7.0 software and was divided into six clades.
Figure 1. Phylogenetic tree of the GRF genes of six species. Protein sequences from A. thaliana, P. equestris, O. sativa, C. goeringii, C. ensifolium, and C. Sinensis. The phylogenetic tree was constructed using MEGA 7.0 software and was divided into six clades.
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Figure 2. The amino acid multiple-sequence alignment results of CgGRF, CeGRF, and CsGRF proteins. (A) The conserved domain of GRFs; (B) the Seqlogo of GRFs.
Figure 2. The amino acid multiple-sequence alignment results of CgGRF, CeGRF, and CsGRF proteins. (A) The conserved domain of GRFs; (B) the Seqlogo of GRFs.
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Figure 3. Analysis of GRF gene structure and conserved motifs in three Cymbidium species. (A) Phylogenetic tree. (B) Conserved motifs.
Figure 3. Analysis of GRF gene structure and conserved motifs in three Cymbidium species. (A) Phylogenetic tree. (B) Conserved motifs.
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Figure 4. The regulatory elements in the promoter region of three Cymbidium species. (A) The cis-acting elements of C. goeringii. (B) The cis-acting elements of C. ensifolium. (C) The cis-acting elements of C. sinense.
Figure 4. The regulatory elements in the promoter region of three Cymbidium species. (A) The cis-acting elements of C. goeringii. (B) The cis-acting elements of C. ensifolium. (C) The cis-acting elements of C. sinense.
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Figure 5. Collinear correlation analysis in three Cymbidium species. (A) Synteny analysis of GRF genes in C. goeringii. (B) Synteny analysis of GRF genes in C. ensifolium. (C) Synteny analysis of GRF genes in C. sinense. The red lines represent the pairs of segmental duplicated gene.
Figure 5. Collinear correlation analysis in three Cymbidium species. (A) Synteny analysis of GRF genes in C. goeringii. (B) Synteny analysis of GRF genes in C. ensifolium. (C) Synteny analysis of GRF genes in C. sinense. The red lines represent the pairs of segmental duplicated gene.
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Figure 6. (A) The expression pattern of CgGRFs in C. goeringii. The heat map of CgGRFs in different parts of C. goeringii. The color scale on the right side of the heatmap represents the relative expression level of CgGRFs, and the expression was increased with the color gradient from orange to purple. The FPKM values of CgGRFs in C. goeringii are listed in Table S4. (B) Analysis of gene expression of six CgGRFs in C. goeringii through real-time quantitative fluorescence. Different letters indicate significant differences between groups based on a one-way analysis of variance (ANOVA) with Duncan’s multiple range test.
Figure 6. (A) The expression pattern of CgGRFs in C. goeringii. The heat map of CgGRFs in different parts of C. goeringii. The color scale on the right side of the heatmap represents the relative expression level of CgGRFs, and the expression was increased with the color gradient from orange to purple. The FPKM values of CgGRFs in C. goeringii are listed in Table S4. (B) Analysis of gene expression of six CgGRFs in C. goeringii through real-time quantitative fluorescence. Different letters indicate significant differences between groups based on a one-way analysis of variance (ANOVA) with Duncan’s multiple range test.
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Table 1. Physicochemical property analysis of the GRF genes in three Cymbidium species.
Table 1. Physicochemical property analysis of the GRF genes in three Cymbidium species.
Gene
Name		Gene IDNumber of
Amino Acids
(AA)		Theoretical
PI		Molecular
Weight
(Mw)		Instability
Index
(II)		Aliphatic
Index
(AI)		Grand Average of Hydropathicity (GRAVY)Subcellular
Localization
CgGRF1GL002403597.240.9261.6557.10−0.669Nucleus
CgGRF2GL000043778.4541.0954.5657.48−0.609Nucleus
CgGRF3GL134101888.9720.5957.1668.56−0.556Nucleus
CgGRF4GL181945457.6160.1769.5264.99−0.602Nucleus
CgGRF5GL187975788.5641.1761.3752.23−0.690Nucleus
CgGRF6GL133025149.1955.4052.5967.98−0.470Nucleus
CgGRF7GL164663299.0436.4361.4463.22−0.516Nucleus
CgGRF8GL169212798.7632.2975.2545.84−0.977Nucleus
CgGRF9GL086264788.8252.3956.5060.42−0.642Nucleus
CgGRF10GL1438529510.0634.0352.0853.66−0.880Nucleus
CgGRF11GL214105848.765.0765.7967.83−0.582Nucleus
CeGRF1JL0198542828.4132.0665.9749.86−0.833Nucleus
CeGRF2JL0000343697.7240.3056.5856.61−0.632Nucleus
CeGRF3JL0092852058.9622.4256.7363.37−0.533Nucleus
CeGRF4JL0207315557.6361.3568.7968.79−0.564Nucleus
CeGRF5JL0119923398.6637.7959.9659.32−0.585Nucleus
CeGRF6JL0092841539.4316.7064.8169.54−0.518Nucleus
CeGRF7JL0237253049.8835.5658.8452.37−1.057Nucleus
CeGRF8JL0075732648.430.5975.4741.06−1.045Nucleus
CeGRF9JL0182393109.6135.7972.7662.90−0.903Nucleus
CeGRF10JL0066675857.3363.7252.2262.36−0.611Nucleus
CeGRF11JL0017345699.0361.0552.6369.44−0.442Nucleus
CsGRF1cymsin_Mol0163053597.1840.9061.2357.10−0.669Nucleus
CsGRF2cymsin_Mol0057543708.4340.3454.2558.57−0.584Nucleus
CsGRF3cymsin_Mol0080351888.9720.5856.9968.03−0.558Nucleus
CsGRF4cymsin_Mol0114925838.7965.1163.8967.77−0.587Nucleus
CsGRF5cymsin_Mol0126503409.0737.8461.4259.15−0.550Nucleus
CsGRF8cymsin_Mol0010742798.7632.2874.8145.84−0.975Nucleus
CsGRF9cymsin_Mol0019203109.4935.8269.2163.87−0.886Nucleus
CsGRF10cymsin_Mol0117814608.5449.9650.2564.89−0.603Nucleus
CsGRF11cymsin_Mol0001795699.1261.1253.0270.30−0.433Nucleus
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Deng, Y.; Pan, Y.; Wang, F.; Chen, F.; Wu, X.; Chen, J.; Zhu, J.; Peng, D. Genome-Wide Identification of the Growth-Regulating Factor (GRF) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii. Horticulturae 2025, 11, 1015. https://doi.org/10.3390/horticulturae11091015

AMA Style

Deng Y, Pan Y, Wang F, Chen F, Wu X, Chen J, Zhu J, Peng D. Genome-Wide Identification of the Growth-Regulating Factor (GRF) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii. Horticulturae. 2025; 11(9):1015. https://doi.org/10.3390/horticulturae11091015

Chicago/Turabian Style

Deng, Yan, Yun Pan, Fei Wang, Feng Chen, Xiaopei Wu, Jinliao Chen, Jin Zhu, and Donghui Peng. 2025. "Genome-Wide Identification of the Growth-Regulating Factor (GRF) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii" Horticulturae 11, no. 9: 1015. https://doi.org/10.3390/horticulturae11091015

APA Style

Deng, Y., Pan, Y., Wang, F., Chen, F., Wu, X., Chen, J., Zhu, J., & Peng, D. (2025). Genome-Wide Identification of the Growth-Regulating Factor (GRF) Gene Family in Three Cymbidium Species and Expression Patterns in C. goeringii. Horticulturae, 11(9), 1015. https://doi.org/10.3390/horticulturae11091015

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