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Article

The Identification of Auxin Response Factors and Expression Analyses of Different Floral Development Stages in Roses

by
Rui Huang
1,2,
Xiaoni Zhang
2,3,
Kaiqing Luo
2,
Luke R. Tembrock
4,
Sen Li
1,* and
Zhiqiang Wu
1,2,3,*
1
College of Horticulture, Shanxi Agricultural University, Jinzhong 030801, China
2
Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China
3
Kunpeng Institute of Modern Agriculture at Foshan, Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, China
4
Department of Agricultural Biology, Colorado State University, Fort Collins, CO 80523, USA
*
Authors to whom correspondence should be addressed.
Genes 2025, 16(1), 41; https://doi.org/10.3390/genes16010041
Submission received: 5 December 2024 / Revised: 25 December 2024 / Accepted: 25 December 2024 / Published: 1 January 2025
(This article belongs to the Section Plant Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
Background/Objectives: Auxin response factors (ARFs) are important in plant growth and development, especially flower development. However, there is limited research on the comprehensive identification and characterization of ARF genes in roses. Methods: We employed bioinformatics tools to identify the ARF genes of roses. These genes were characterized for their phylogenetic relationships, chromosomal positions, conserved motifs, gene structures, and expression patterns. Results: In this study, a total of 17 ARF genes were identified in the genomes of Rosa chinensis ‘OB’, R. chinensis ‘CH’, R. rugosa, and R. wichurana. Based on RNA-seq analyses, we found that the ARF genes had diverse transcript patterns in various tissues and cultivars. In ‘CH’, the expression levels of RcCH_ARFs during different flower-development stages were classified into four clusters. In cluster 3 and cluster 4, RcCH_ARFs were specifically high and low in different stages of floral evocation. Gene expression and phylogenetic analyses showed that RcCH_ARF3, RcCH_ARF4, and RcCH_ARF18 were likely to be the key genes for rose flower development. Conclusions: The identification and characterization of ARF genes in Rosa were investigated. The results presented here provide a theoretical basis for the molecular mechanisms of ARF genes in plant development and flowering for roses, with a broader application for other species in the rose family and for the development of novel cultivars.

1. Introduction

Auxin is a crucial phytohormone that has various effects on the regulators of plant growth and development [1]. There are several key auxin response genes, such as auxin response factors, that play central roles in the auxin response pathway of plants [2,3]. ARFs can mediate auxin transcriptional regulation by binding to auxin-responsive elements (TGTCTC, TGTCCC, TGTCAC, and TGTCGG) at the promoters of auxin-responsive genes [4,5]. ARFs typically contain the following three domains: a B3-like DNA-binding domain (DBD) at the N-terminus that specifically recognizes and binds the auxin response element; a middle domain that plays an activating or inhibiting role; and a C-terminus dimerization domain (CTD) that can bind directly to auxin/indole-3-acetic acid (Aux/IAA) [6,7]. ARFs participate in the auxin regulatory pathway in plants by binding to Aux/IAA [8]. When auxin concentrations are low, ARFs bind to Aux/IAA to form a dimer, which then binds to transcriptional repressors to prevent binding to target genes, thereby inhibiting the expression of auxin-responsive genes [9,10]. Conversely, when auxin concentrations are elevated, ARFs are released and the inhibitory effect is eliminated, leading to a series of auxin response processes [11,12].
Arabidopsis thaliana is the most extensively and comprehensively studied plant species in which the ARF gene family has been described and contains 23 AtARF members [13]. Researchers have elucidated the functions of AtARFs, providing detailed references for ARF gene family studies in other plant species. For example, AtARF1 and AtARF2 affect leaf senescence and floral organ shedding [14]. AtARF3 regulates shoot apical meristem maintenance [15]. AtARF19 and AtARF7 control lateral root growth [16]. Furthermore, ARFs are also involved in flower organ growth: arf6 and arf8 mutants showed a slight delay in stem elongation and floral organ growth in A. thaliana [17]. AtARF3 regulates the determinacy of the floral meristem [18]. AtARF2–AtARF4 and AtARF5 have essential roles in regulating both female and male gametophyte development [19]. Additionally, ARF genes have been comprehensively identified in various plants, including 23 in Oryza sativa [20], 31 in Zea mays [21], 22 in Solanum lycopersicum [22], 24 in Cicer arietinum [23], 20 in Solanum melongena [24], 13 in Pinus koraiensis [25], and 26 in Dendrobium officinale [26]. To date, there has not been a comprehensive study of ARF genes in roses; thus, it is necessary to identify and analyze ARF genes in roses to better understand their role in flowering.
Roses are one of the most famous flowers in the world, due in large part to their diverse, beautiful, and long-lasting flowers. Rose flowers are widely grown for use as cut flowers and as garden ornamentals, with extremely high esthetic and economic values. Auxin is a key hormone in rose development [27,28] and studies have demonstrated the essential role of auxin in the process of growth in roses. For instance, exogenous indole-3-acetic acid and naphthalene acetic acid affected the regeneration of damask-rose cuttings in three different growth media [29]. Auxin–cytokinin homeostasis is involved in the adventitious root formation of rose cuttings [30]. Petal abscission in fragrant roses is associated with auxin pathways [31]. As important genes in the auxin regulatory pathway, ARFs have been the subject of research regarding their function in the flower development of roses. For example, the RhARF7–RhSUC2 module has been shown to be involved in the auxin regulation of SUC transport and, subsequently, regulates petal shedding [32]. Alternatively, RhARF18 recruits RhHDA6 to the promoter of RhAG to control petal–stamen transformation by enhancing inhibitory effects through histone deacetylation [33]. Furthermore, RhARF2 regulates flower opening by controlling RhMYB6 expression and by mediating the crosstalk between auxin and ethylene signaling [34]. However, the regulation of auxin on flower development in roses is minimal and the comprehensive identification of ARF genes in roses is still lacking.
With the completion of genome identification for several Rosa species and cultivars, it is possible to analyze genes that play an important role in flower growth and development. In this study, 17 ARF members were identified in four Rosa genomes. We analyzed the physical and chemical properties as well as the homology, conserved motifs, and gene structure for each gene. Furthermore, the expressions of these genes in different tissues and during different flower-development stages were investigated to understand the important roles of ARFs in Rosa. The results of this study provide a new foundation to further study the function of ARFs in rose flower development specifically as well as plant growth more generally.

2. Materials and Methods

2.1. Identification and Sequence Analysis of ARF Genes in Roses

The genomes of R. chinensis ‘Old Blush’ (hereafter ‘OB’) [35], R. chinensis ‘Chilong Hanzhu’ (hereafter ‘CH’) [36], R. rugosa [37], and R. wichurana [38] were downloaded from the Genome Database for Rosaceae (http://www.rosaceae.org/, accessed on 6 May 2024) and the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/, accessed on 6 May 2024) (accession number: PRJNA932466). The 23 ARF amino acid sequences of A. thaliana [13] and 25 ARF amino acid sequences of O. sativa [20] were obtained from The Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org/, accessed on 6 May 2024) and the Rice Genome Annotation Project (RGAP; https://rice.uga.edu/, accessed on 6 May 2024) as query sequences, respectively. Then, the ARFs of the roses were identified as follows: first, ARF amino acid sequences from A. thaliana and O. sativa were used as queries to search for ARF genes in R. chinensis ‘OB’, R. chinensis ‘CH’, R. rugosa, and R. wichurana via a local BLASTP, with an E-value of ≤10−5. The sequences of ARFs were further confirmed by using the hidden Markov model (HMM) file (PF06507) from the Pfam database (http://pfam.xfam.org/search, accessed on 6 May 2024). The NCBI conserved domain database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 6 May 2024) and SMART (https://smart.embl.de/, accessed 6 May 2024) were used to detect the amino acid sequences of candidate ARF members in Rosa. After identifying ARF members, ExPASy (http://web.expasy.org/protparam/, accessed on 7 May 2024) was used to analyze the physicochemical properties, such as the number of amino acids and molecular formulae. A subcellular localization prediction was conducted using Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 7 May 2024).

2.2. Phylogenetic Analysis of ARFs in Roses

The full-length amino acid sequences of ARFs in A. thaliana, O. sativa, Fragaria vesca [39], and four Rosa cultivars were used to construct a phylogenetic tree by using the neighbor-joining method with 1000 iterations to obtain bootstrap values to assess the clade support. The phylogenetic tree was constructed using MEGA11 [40]. The tree was then visualized using the online tool Evolview v3 [41].

2.3. Chromosomal Location Analysis of ARFs

Using TBtools [42], we obtained the chromosome locations for each ARF gene according to the genome annotation file of the four Rosa cultivars.

2.4. Motifs and Gene Structure Analysis of ARFs

MEME (https://meme-suite.org/meme/tools/meme, accessed 12 May 2024) was used to identify the conserved motifs in the ARF amino acid sequences from the four Rosa cultivars using the patterns of 15 motifs. The gene structure information of the ARFs was confirmed using annotation data from the four genomes. TBtools was used to visualize the results.

2.5. Transcriptome Analysis of ARFs

To compare the expression patterns of ARFs, RNA-seq data from different tissues of R. chinensis ‘OB’ [43] and R. chinensis ‘CH’ [36] were downloaded. The transcriptome data of ‘CH’ samples during different flower-development stages were also used. The flower-development stages of ‘CH’ were divided into seven stages, including the vegetative meristem stage (DBO), the initiation stages of petals/petal-like structures and stamens/stamen-like structures (ISPS), the stage in which the hypanthium starts to sink below the perianth and stamens (HBPS), the early stage of flower bud formation (FBGP), the development of noncolored petals of flower buds (noncolored), the coloration of petals before flowering (coloring), and the emergence of fully colored petals during flower blooming (colored). Clean reads were obtained by removing low-quality reads and adapter sequences from the raw data. Using Hisat2 2.1.0 software, we mapped the clean reads to the reference genome [35,36]. The output of mapping was processed using String Tie to obtain FPKM (fragments per kilobase of exon model per million mapped fragments). The statistical analyses were conducted using GraphPad Prism and heat maps were drawn using TBtools.

3. Results

3.1. Identification of ARF Genes in Four Rosa Cultivars

To identify the ARFs of roses, local BLASTP and HMM searches were performed and domain detections were conducted. Finally, from each of four Rosa cultivars (R. chinensis ‘OB’, R. chinensis ‘CH’, R. rugosa, and R. wichurana), we identified 17 ARF members. The ARFs in the Rosa species were renamed based on homologous relationships with F. vesca (Table 1). Then, in order to more clearly understand the characteristics of ARFs in the Rosa species, the number of amino acids, molecular formula, prediction of molecular weight, theoretical isoelectric points, instability index, and prediction of subcellular localization were analyzed (Table S1). The number of amino acids of the ARFs ranged from 608 (RcOB_ARF17) to 1163 (RcOB_ARF7) in ‘OB’, 606 (RcCH_ARF17) to 1160 (RcCH_ARF7) in ‘CH’, 607 (RrARF17) to 1181 (RrARF7) in R. rugosa, and 569 (RwARF17) to 1165 (RwARF7) in R. wichurana. The predicted molecular weights for the ARF proteins ranged from 66.55 (RcOB_ARF17) to 130.21 (RcCH_ARF7), 66.36 (RcCH_ARF17) to 129.75 (RcCH_ARF7), 66.26 (RrARF17) to 1332.22 (RrARF7), and 62.68 (RwARF17) to 130.25 (RwARF7) kDa in ‘OB’, ‘CH’, R. rugosa, and R. wichurana, respectively. The theoretical isoelectric points varied from 5.22 (RcOB_ARF5) to 7.93 (RcOB_ARF18), 5.28 (RcCH_ARF5) to 7.92 (RcCH_ARF18), 5.21 (RrARF5) to 7.90 (RrARF18), and 5.22 (RwARF5) to 7.61 (RwARF18) in ‘OB’, ‘CH’, R. rugosa, and R. wichurana, respectively. The prediction of subcellular localization in the four Rosa cultivars showed that all genes were located in the nucleus.

3.2. Phylogenetic Analysis

To analyze the evolutionary relationship of ARF genes in roses, a phylogenetic tree was constructed using 134 full-length amino acid sequences of ARFs from A. thaliana, O. sativa, F. vesca, and four Rosa cultivars (Figure 1). The 134 sequences were grouped into the following four major groups: Group I, Group II, Group III, and Group IV. Group I was further subdivided into Group IA, Group IB, and Group IC. Among these groups, Group II contained the greatest number of ARF genes in all four Rosa cultivars, which contained 6, 6, 6, and 5 ARFs in ‘OB’, ‘CH’, R. rugosa, and R. wichurana, respectively. Group III had the fewest members, with only two members in each cultivar of Rosa. A further analysis found that all ARFs of ‘OB’, ‘CH’, R. rugosa, R. wichurana, and FveARFs were on adjacent branches of the evolutionary tree, indicating the expected phylogenetic relationship of these genes as they are all species in the Rosaceae family.

3.3. Chromosomal Locations of ARFs in Rosa

We further investigated the chromosomal locations of ARF genes in Rosa (Figure 2). The analysis showed that RcOB_ARFs and RcCH_ARFs were unevenly distributed on 7 chromosomes, RrARFs were distributed across 6 of the 7 chromosomes, and RwARFs were distributed on 7 chromosomes and a contig. The largest number of ARF genes were found on chromosome 5 (Chr5) and Chr7, each with four genes. Chr4 of ‘OB’ and ‘CH’ contained the least number of RcOB_ARFs and RcCH_ARFs, with only one gene (comprising RcOB_ARF19 and RcCH_ARF19). Chr3, Chr4, and Contig00606 of R. wichurana also contained only one gene, comprising RwARF18-like1, RwARF9, and RwARF17-like, respectively. Chr1 of R. rugosa, which was homologous with Chr4 of the other three Rosa cultivars, had no ARF genes. Moreover, Chr6 of R. rugosa had one more ARF than the homologous chromosomes of the other three Rosa cultivars, with three genes. A collinearity analysis showed that the ARF gene family of ‘OB’, ‘CH’, and R. wichurana had four pairs of segmental repeats, while R. rugosa had only three pairs of segmental repetitive genes (Figure S1).

3.4. Motifs and Gene Structure Analysis of the Four Rosa Cultivars

To study the characteristic regions of ARF proteins in roses, 15 conserved motifs in rose ARF genes were identified using MEME and then analyzed in conjunction with the evolutionary tree (Figure 3A,B and Figure S2). Among them, motifs 2, 1, and 9 were identified as the B3-like DNA-binding domain (DBD) located at the N-terminus. Motifs 8, 12, 6, 7, and 11 represented the Auxin_resp domain. Motifs 10, 4, and 14 represented dimerization domains at the C-terminus (namely, CTD). The results showed that most ARF genes contained all three domains, while some members of Group III and Group IV only had the B3 and Auxin_resp domains and did not contain CTDs (ARF3, ARF17, ARF17-like, and ARF18-like2). The proportion of ARF members lacking the CTD was 17.6%, 17.6%, 17.6%, and 23.5% in ‘OB’, ‘CH’, R. rugosa, and R. wichurana, respectively. A further analysis found that the numbers and distribution of motifs were similar to their corresponding phylogenetic relationships. For example, the genes from Group I all had 13 or 14 motifs and were similar in structure. Similarly, the ARFs of Group II all contained 14 motifs and were similar in structure.
To explore the structural arrangement of ARF genes in roses, the exons and introns were analyzed (Figure 3C). The results showed that the distribution and number of exons in the ARF genes on the same branches were similar. For example, the ARFs from Group III contained 11 or 12 exons and their positions were similar. Additionally, the ARF genes in Group IV had the fewest exons, with only 2-5; their arrangements were also similar. The motifs and gene structure analyses both indicated that ARFs were highly conserved within a lineage, which helped us to further analyze the phylogenetic relationships and regulatory function of rose ARFs.

3.5. Expression of ARFs in Roses

Studies have shown that ARFs are involved in the growth and development of roots, stems, leaves, and flowers [6,7,44]. In order to better understand the potential function of ARFs in different tissues of roses, the expression levels of ARFs in different tissues of ‘OB’ and ‘CH’ were analyzed (Figure 4). We found that there were differences in the expression of ARFs between the two cultivars of roses; some genes showed a tissue-specific high expression, which may have been related to their function. For example, in ‘OB’, RcOB_ARF5 was particularly highly expressed in the roots compared with the other tissues. However, in ‘CH’, the expression level of the homologous gene RcCH_ARF5 was not only higher in the roots than in the other tissues, but also the expression level was significantly higher in the flowers than in the stems and leaves. Furthermore, in ‘CH’, RcCH_ARF3 had a higher expression level in the stems compared with the other three tissues. However, in ‘OB’, the expression in the roots, stems, and leaves of homologous gene RcOB_ARF3 was similar and was higher in all these organs than in the flowers. Additionally, the expression level of RcOB_ARF8 in ‘OB’ was significantly higher in the flowers than in the other tissues and a similar pattern was observed for RcOB_ARF6 and RcOB_ARF6-like. In ‘CH’, not only did the homologous genes RcCH_ARF8, RcCH_ARF6, and RcCH_ARF6-like possess similar expression patterns (as mentioned above), but also RcCH_ARF18-like3 and RcCH_ARF2 showed similar expression levels.
To more deeply investigate the role of ARFs in rose flower development, the expressions of RcCH_ARF genes during different stages of flower development were analyzed (Figure 5). The results showed that the expression patterns of RcCH_ARFs were classified into four clusters, which may be involved in different flower-development stages. The genes of cluster 1 had a low expression during all stages. The expression of RcCH_ARFs in cluster 2 were high throughout all flower-development stages. The expression of RcCH_ARF genes in cluster 3 and cluster 4 fluctuated during flower-development stages and showed similar expression trends, respectively. For instance, RcCH_ARF4 in cluster 3 was highly expressed during the first three stages (DBO, ISPS, and HBPS), with a lower expression in later stages (FBGP, noncolored, coloring, and colored). Furthermore, the expression level of RcCH_ARF18 in cluster 4 was low during the DBO–HBPS stages, but increased during FBGP through to the colored stage. Previous research has reported that FveARF4 (homologous to RcCH_ARF4), AtARF3 (homologous to RcCH_ARF3), and FveARF18A (homologous to RcCH_ARF18) are all involved in the regulation of flower development [18,39,45]. Thus, it could be inferred that RcCH_ARF4, RcCH_ARF3, and RcCH_ARF18 may have similar regulatory roles during the stages of flower development.

4. Discussion

Auxin is an essential phytohormone involved in all stages of plant growth and development, including organogenesis, tissue differentiation, root initiation, fruit development, seed growth, and especially in the development of flowers [46,47,48,49]. These biological processes cannot be initiated without auxin. As important transcriptional factors, ARFs are involved in the auxin signal transduction pathway and play pivotal roles in regulating the expression of auxin-responsive genes [50,51]. For example, FveARF2 can negatively regulate strawberry-fruit ripening and quality [52]. FaARF2 mediates the receptacle ripening of strawberries via auxin–ABA interplay [53]. In addition, the Mdm-miR160–MdARF17–MdWRKY33 module regulates cold stress tolerance by mediating ROS scavenging upon cold exposure in apples [54]. Auxin induces ethylene biosynthesis in apple fruit by activating the expression of MdARF5, which initiates apple-fruit ripening [55]. The positive feedback loop of Mdm-miR160–MdARF17–MdHYL1 is necessary for apple survival under drought stress [56]. PpARF6 plays a positive regulatory role in peach-fruit ripening by integrating auxin and ethylene signaling [57]. There are now numerous studies on fruit ripening and responses to stress, but research on the regulation of flower development by ARFs is relatively scarce.
Roses have become one of the most popular flowers in the world because of their rich color, fragrant aroma, and other desirable traits. Therefore, the analysis and characterization of ARF genes in Rosa species is necessary and can deepen our understanding of the role of ARF genes in rose development and aid in the development of new cultivars. In this study, based on the published genomes from four Rosa cultivars, a total of 17 ARFs were identified in ‘OB’, ‘CH’, R. rugosa, and R. wichurana, respectively. The genome sizes of these cultivars were 515 Mb [35], 541 Mb [36], 382 Mb [37], and 530 Mb [38] for ‘OB’, ‘CH’, R. rugosa, and R. wichurana, respectively. Among other plant genomes that have been sequenced and analyzed for ARF genes, A. thaliana (125 Mb) contains 23 AtARFs [13], O. sativa (466 Mb) contains 25 OsARFs [20], Z. mays (2.3 Gb) contains 31 ZmARFs [21], and Osmanthus fragrans (727 Mb) contains 50 OfARFs [58]. The results above show that there is no association between the number of ARF genes and the genome size among different species.
The identification of conserved motifs is the foundation for studying gene functions. Our analysis of motifs found that ARFs on the same branch of the evolutionary tree had similar numbers and structural arrangements of motifs. Our results showed that all genes contained two structural domains, which were DBD and Auxin_resp domains. However, the CTDs of ARF3, ARF17, ARF17-like, and ARF18-like2 were truncated. The proportion of ARF genes lacking the CTD in Rosa species (truncated RcOB_ARFs accounted for 17.6%, RcCH_ARFs for 17.6%, RrARFs for 17.6%, and RwARFs for 23.5%) were lower compared with other species such as Medicago truncatula (37.5%) [59], Linum usitatissimum (42.4%) [60], and P. koraiensis (38.5%) [25]. As such, it could be inferred that the CTD may be a key structural domain of ARF genes in Rosa species.
Phylogenetic trees are important methods used to resolve homologous relationships among various species or genes. In this study, we found that the ARF genes of the four Rosa cultivars we studied were most closely related to FveARFs, which reflected the taxonomic similarity of the lineages as they are both in the Rosaceae family. The function of ARFs has been extensively studied in various species, providing a valuable reference for exploring the potential functions of Rosa ARF genes. For example, the FvemiR160–FveARF18A–FveAP1/FveFUL module was verified to regulate the flowering time of woodland strawberries [39]. In Rosa, RcOB_ARF18, RcCH_ARF18, RrARF18, and RwARF18 were genetically most similar to FveARF18A. Therefore, we could infer that ARF18 of the Rosa species might play a similar role in regulating flower development. Additionally, the miR390–tasiRNA3–ARF4 pathway delays the flowering time of woodland strawberries through FveAP1/FveFUL [45]. ARF4 in the four Rosa cultivars was homologous to FveARF4, so it is reasonable to presume that ARF4 of roses may function in a similar regulatory role in flower development. The function of ARFs in the model species A. thaliana has also been well-studied; for example, AtARF3, which controls floral meristem maintenance and termination by regulating cytokinin biosynthesis and signaling [61]. The miR172/AP2 module regulates the size of the inflorescence meristem and AP2 is regulated by ARF3, controlling the determination of the SAM size in A. thaliana [62]. Thus, ARF3 in Rosa species may also regulate flower development. These comparisons of Rosa ARFs with other species provide an important set of hypotheses to test in future work.
Gene expression is an important criterion for determining if a gene is functioning in a given tissue or at time of development. In this study, we discovered that some genes showed flower-specific high expression; these were RcOB_ARF8, RcOB_ARF6, and RcOB_ARF6-like in ‘OB’ and RcCH_ARF18-like3 in ‘CH’. We also found that RcCH_ARF2 in ‘CH’ was not specifically highly expressed in flowers, but its expression level in flowers was higher than in other tissues. The expression patterns of RcCH_ARF8, RcCH_ARF6, RcCH_ARF6-like, and RcCH_ARF4 in ‘CH’ were similar to RcCH_ARF2. Interestingly, of all the genes mentioned above in ‘CH’, RcCH_ARF4 had high expression levels during the first three stages of floral evocation (DBO, ISPS, and HBPS), but the expression levels decreased during subsequent stages. In F. vesca, the function of the homologous gene FveARF4 has been confirmed in flower development [45]. Therefore, RcCH_ARF4 may have a downregulating role in the flower development of roses. This inference requires experiments to further demonstrate the mechanism of RcCH_ARF4. These results provide important data and well-resolved targets to help verify the function of ARF genes in regulating flower development. Knowing how ARF genes function in detail allows for the selection of desirable gene variants for the development of improved cultivars.
Research has reported the functions of ARFs in flower development [32,33,34], but there is still a limitation of genome-wide analyses and the characterization of ARF genes in roses. Moreover, research on the impact of ARFs on flower development in roses is scarce. Here, we conducted a comprehensive identification and bioinformatics analysis of ARFs in Rosa. Significantly, we identified ARF genes that may be involved in the regulation of flower development in roses; these were RcCH_ARF3, RcCH_ARF4, and RcCH_ARF18. From these analyses, this study aimed to comprehensively understand ARF genes in roses and reveal their roles in regulating flower development. These results provide information for the further functional analysis of ARF genes in roses, ultimately improving floral developmental traits in roses.

5. Conclusions

In this study, we performed a genome-wide identification of ARF genes in four Rosa cultivars, and a total of 17 ARF genes were identified in ‘OB’, ‘CH’, R. rugosa, and R. wichurana, respectively. These ARF genes were unevenly distributed on 7 chromosomes. An evolutionary analysis divided the ARFs of roses into four groups according to their homology with AtARFs, OsARFs, and FveARFs. The conserved motifs and gene structure analysis suggested that genes from the same lineage represented similar structures. The gene expression showed that ARF genes had different expression patterns in various tissues of ‘OB’ and ‘CH’. In ‘CH’, the expressions of RcCH_ARFs in cluster 3 and cluster 4 fluctuated during the flower-development stages, which were specifically high and low in the floral evocation stages, indicating that the ARF genes of roses may play essential roles in flower development. Importantly, RcCH_ARF3, RcCH_ARF4, and RcCH_ARF18 were identified as highly likely to be regulatory factors in the flower development of roses. In short, this study provides valuable information for the functional verification of ARFs in rose flower development, which is applicable to future studies of functional genomics and the development of new cultivars.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/genes16010041/s1. Figure S1: The chromosome distribution and interchromosomal relationships of ARF genes in four Rosa cultivars. (A) The chromosome distribution and interchromosomal relationships of RcOB_ARFs in ‘OB’. (B) The chromosome distribution and interchromosomal relationships of RcCH_ARFs in ‘CH’. (C) The chromosome distribution and interchromosomal relationships of RrARFs in R. rugosa. (D) The chromosome distribution and interchromosomal relationships of RwARFs in R. wichurana. Figure S2: The sequence logos of 15 conserved protein motifs in ARF genes of four Rosa cultivars. Table S1: Physicochemical properties of ARFs in four Rosa species.

Author Contributions

Conceptualization, Z.W. and S.L.; methodology, R.H. and X.Z.; software, R.H.; validation, R.H. and X.Z.; formal analysis, R.H.; investigation, R.H., X.Z. and K.L.; resources, Z.W. and S.L.; data curation, R.H.; writing—original draft preparation, R.H.; writing—review and editing, R.H., X.Z., K.L., L.R.T., S.L. and Z.W.; visualization, R.H.; supervision, Z.W. and S.L.; project administration, Z.W. and S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by funding from the Funding of Major Scientific Research Tasks, Kunpeng Institute of Modern Agriculture at Foshan (ZXFR2024004) to X.Z. This work was also supported by the Shenzhen Fundamental Research Program (Grant No. JCYJ20220818103212025) and the Chinese Academy of Agricultural Sciences Elite Youth Program (110243160001007) to Z.W.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Genes 16 00041 g001
Figure 1. Phylogenetic tree of ARFs. Different-colored arcs represent different groups. Five stars of different colors represent different Rosa species (R. chinensis ‘OB’ in red, R. chinensis ‘CH’ in blue, R. rugosa in green, and R. wichurana in yellow). The lavender circles represent F. vesca, the light-pink triangles represent A. thaliana, and the light-blue squares represent O. sativa.
Figure 1. Phylogenetic tree of ARFs. Different-colored arcs represent different groups. Five stars of different colors represent different Rosa species (R. chinensis ‘OB’ in red, R. chinensis ‘CH’ in blue, R. rugosa in green, and R. wichurana in yellow). The lavender circles represent F. vesca, the light-pink triangles represent A. thaliana, and the light-blue squares represent O. sativa.
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Figure 2. Chromosomal locations of ARF genes in four Rosa cultivars. (A) Chromosome location of RcOB_ARFs. (B) Chromosome location of RcCH_ARFs. (C) Chromosome location of RrARFs. (D) Chromosome location of RwARFs. Chromosome numbers are shown to the left of each chromosome. The scale on the left is chromosomal length in Mb.
Figure 2. Chromosomal locations of ARF genes in four Rosa cultivars. (A) Chromosome location of RcOB_ARFs. (B) Chromosome location of RcCH_ARFs. (C) Chromosome location of RrARFs. (D) Chromosome location of RwARFs. Chromosome numbers are shown to the left of each chromosome. The scale on the left is chromosomal length in Mb.
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Genes 16 00041 g003
Figure 3. Phylogenetic tree, motifs, and gene structure of ARF genes in four Rosa cultivars. (A) Phylogenetic relationships of ARF genes from four rose cultivars. The pink-, purple-, yellow-, blue-, and green-colored blocks on the tree denote Group IA, Group IC, Group II, Group III, and Group IV, respectively. (B) Motif analyses of ARFs. The colored boxes, numbered 1–15, indicate different conserved protein motifs. (C) Gene structure of ARF genes. Exons are represented by yellow boxes; the lines between the boxes are introns.
Figure 3. Phylogenetic tree, motifs, and gene structure of ARF genes in four Rosa cultivars. (A) Phylogenetic relationships of ARF genes from four rose cultivars. The pink-, purple-, yellow-, blue-, and green-colored blocks on the tree denote Group IA, Group IC, Group II, Group III, and Group IV, respectively. (B) Motif analyses of ARFs. The colored boxes, numbered 1–15, indicate different conserved protein motifs. (C) Gene structure of ARF genes. Exons are represented by yellow boxes; the lines between the boxes are introns.
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Figure 4. RcARF gene expression patterns in different tissues. (A) RcOB_ARFs expression in different tissues. (B) RcCH_ARFs expression in different tissues. The bars indicate one SD. Expression levels are given in FPKM.
Figure 4. RcARF gene expression patterns in different tissues. (A) RcOB_ARFs expression in different tissues. (B) RcCH_ARFs expression in different tissues. The bars indicate one SD. Expression levels are given in FPKM.
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Figure 5. Expression of RcCH_ARFs during different stages of flower development. (A) Schematic diagram of different flower-development stages in R. chinensis ‘CH’. (B) Expression of RcCH_ARFs during different flower-development stages in R. chinensis ‘CH’. The results shown in the diagram are log2 (FPKM).
Figure 5. Expression of RcCH_ARFs during different stages of flower development. (A) Schematic diagram of different flower-development stages in R. chinensis ‘CH’. (B) Expression of RcCH_ARFs during different flower-development stages in R. chinensis ‘CH’. The results shown in the diagram are log2 (FPKM).
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Table 1. Gene IDs and gene names of ARFs in Rosa cultivars.
Table 1. Gene IDs and gene names of ARFs in Rosa cultivars.
SpeciesGene NameGene ID
R. chinensis ‘OB’RcOB_ARF18-like3RcHm_v2.0_Chr1g0360041
RcOB_ARF9RcHm_v2.0_Chr7g0186081
RcOB_ARF1RcHm_v2.0_Chr7g0219771
RcOB_ARF2RcHm_v2.0_Chr7g0188911
RcOB_ARF2-likeRcHm_v2.0_Chr6g0292551
RcOB_ARF5RcHm_v2.0_Chr6g0302551
RcOB_ARF7RcHm_v2.0_Chr2g0095551
RcOB_ARF19RcHm_v2.0_Chr4g0397771
RcOB_ARF8RcHm_v2.0_Chr3g0487771
RcOB_ARF6RcHm_v2.0_Chr5g0014961
RcOB_ARF6-likeRcHm_v2.0_Chr2g0152951
RcOB_ARF4RcHm_v2.0_Chr5g0060011
RcOB_ARF3RcHm_v2.0_Chr5g0009381
RcOB_ARF17RcHm_v2.0_Chr7g0240691
RcOB_ARF18RcHm_v2.0_Chr5g0058761
RcOB_ARF18-like1RcHm_v2.0_Chr3g0469661
RcOB_ARF18-like2RcHm_v2.0_Chr1g0363241
R. chinensis ‘CH’RcCH_ARF18-like3evm.model.hB_v1.0_chr1.2384
RcCH_ARF9evm.model.hB_v1.0_chr7.760
RcCH_ARF1evm.model.hB_v1.0_chr7.3082
RcCH_ARF2evm.model.hB_v1.0_chr7.966
RcCH_ARF2-likeevm.model.hB_v1.0_chr6.2903
RcCH_ARF5evm.model.hB_v1.0_chr6.3683
RcCH_ARF7evm.model.hB_v1.0_chr2.914
RcCH_ARF19evm.model.hB_v1.0_chr4.708
RcCH_ARF8evm.model.hB_v1.0_chr3.2842
RcCH_ARF6evm.model.hB_v1.0_chr5.1066
RcCH_ARF6-likeevm.model.hB_v1.0_chr2.4774
RcCH_ARF4evm.model.hB_v1.0_chr5.3774
RcCH_ARF3evm.model.hB_v1.0_chr5.662
RcCH_ARF17evm.model.hB_v1.0_chr7.4543
RcCH_ARF18evm.model.hB_v1.0_chr5.3710
RcCH_ARF18-like1evm.model.hB_v1.0_chr3.1581
RcCH_ARF18-like2evm.model.hB_v1.0_chr1.2612
R. rugosaRrARF18-like3evm.model.Chr3.3276
RrARF9evm.model.Chr7.749
RrARF1evm.model.Chr7.3607
RrARF2evm.model.Chr7.969
RrARF2-likeevm.model.Chr2.1948
RrARF5evm.model.Chr2.923
RrARF7evm.model.Chr6.1049
RrARF8evm.model.Chr4.3418
RrARF6evm.model.Chr5.5782
RrARF6-like1evm.model.Chr6.5248
RrARF6-like2evm.model.Chr6.5260
RrARF4evm.model.Chr5.2304
RrARF3evm.model.Chr5.6284
RrARF17evm.model.Chr7.5673
RrARF18evm.model.Chr5.2402
RrARF18-like1evm.model.Chr4.1801
RrARF18-like2evm.model.Chr3.3560
R. wichuranaRwARF18-like3Rw1G025590.1
RwARF9Rw7G006530.1
RwARF1Rw7G027920.1
RwARF2Rw7G008560.1
RwARF2-likeRw6G029710.1
RwARF5Rw6G037050.1
RwARF7Rw2G007600.1
RwARF19Rw4G006600.1
RwARF6Rw5G010120.1
RwARF6-likeRw2G040900.1
RwARF4Rw5G036880.1
RwARF3Rw5G006830.1
RwARF17-likeRw0G013480.1
RwARF17Rw7G041690.1
RwARF18Rw5G036140.1
RwARF18-like1Rw3G014400.1
RwARF18-like2Rw1G027880.1
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Huang, R.; Zhang, X.; Luo, K.; Tembrock, L.R.; Li, S.; Wu, Z. The Identification of Auxin Response Factors and Expression Analyses of Different Floral Development Stages in Roses. Genes 2025, 16, 41. https://doi.org/10.3390/genes16010041

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Huang R, Zhang X, Luo K, Tembrock LR, Li S, Wu Z. The Identification of Auxin Response Factors and Expression Analyses of Different Floral Development Stages in Roses. Genes. 2025; 16(1):41. https://doi.org/10.3390/genes16010041

Chicago/Turabian Style

Huang, Rui, Xiaoni Zhang, Kaiqing Luo, Luke R. Tembrock, Sen Li, and Zhiqiang Wu. 2025. "The Identification of Auxin Response Factors and Expression Analyses of Different Floral Development Stages in Roses" Genes 16, no. 1: 41. https://doi.org/10.3390/genes16010041

APA Style

Huang, R., Zhang, X., Luo, K., Tembrock, L. R., Li, S., & Wu, Z. (2025). The Identification of Auxin Response Factors and Expression Analyses of Different Floral Development Stages in Roses. Genes, 16(1), 41. https://doi.org/10.3390/genes16010041

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