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Article

Genome-Wide Identification and Analysis of the Aux/IAA Gene Family in Rosa hybrida—“The Fairy”: Evidence for the Role of RhIAA25 in Adventitious Root Development

by
Wuhua Zhang
1,2,†,
Yifei Zhang
1,2,†,
Minge Huangfu
1,2,
Yingdong Fan
1,2,
Jinzhu Zhang
1,2,
Tao Yang
1,2,
Daidi Che
1,2 and
Jie Dong
1,2,*
1
College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin 150030, China
2
Key Laboratory of Cold Region Landscape Plants and Applications, Harbin 150030, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2024, 14(9), 2005; https://doi.org/10.3390/agronomy14092005
Submission received: 17 June 2024 / Revised: 28 August 2024 / Accepted: 29 August 2024 / Published: 2 September 2024
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figures
Versions Notes

Abstract

:
Propagation of cuttings is the primary method of rose multiplication. Aux/IAA, early response genes to auxin, play an important role in regulating the process of adventitious root formation in plants. However, systematic research on the identification of RhAux/IAA genes and their role in adventitious root formation in roses is lacking. In this study, 34 RhAux/IAA genes were identified by screening the rose genome, distributed on seven chromosomes, and classified into three clades based on the evolutionary tree. An analysis of the cis-acting elements in the promoters of RhAux/IAA genes revealed the presence of numerous elements related to plant hormones, the light signal response, the growth and development of plants, and abiotic stress. RNA-seq analysis identified a key RhIAA25 gene that may play an important role in the generation of adventitious roots in roses. Subcellular localization, yeast self-activation, and tissue-specific expression experiments indicated that RhIAA25 encoded a nuclear protein, had no yeast self-activated activity, and was highly expressed in the stem. The overexpression of RhIAA25 promoted the elongation of the primary root in Arabidopsis but inhibited adventitious root formation. This study systematically identified and analyzed the RhAux/IAA gene family and identified a key gene, RhIAA25, that regulates adventitious root generation in roses. This study offers a valuable genetic resource for investigating the regulatory mechanism of adventitious root formation in roses.

1. Introduction

Roots play an important role in the whole process of plant growth and development. The root is not only an important organ for plants to absorb water and nutrients but also plays an important role in anchoring plants in soil [1,2]. Based on the different locations of root emergence, plant roots can be classified into the following three types: primary roots, adventitious roots, and lateral roots [3]. The formation and development of adventitious roots are vital for the successful propagation of superior germplasm resources and the preservation of elite genes, and they play a key role in plant responses to abiotic stress [4,5]. Adventitious roots are formed from non-root tissues that develop postembryonically from other tissues of the plant. The process of adventitious root formation is divided into the following three main stages: first, the cells undergo reprogramming, which leads to the re-differentiation of the adventitious root, initiating cells to form procambium cells; then, the procambium cells undergo successive divisions to form adventitious root primordia; and finally, adventitious root primordia begin to appear and elongate to form adventitious roots [4,5]. Adventitious rooting is an extremely complex process controlled by a combination of exogenous factors, such as light, water and mineral nutrients, and plant hormones [6,7].
Auxin plays a key role in the induction of adventitious roots and the formation of root morphology in plants [8]. Research indicates that auxin is a core hormone that promotes the formation of adventitious roots in many woody plants [9]. Auxin signal transduction is mainly controlled by two gene families, ARF and Aux/IAA [10]. The Aux/IAA gene family can respond early to auxin induction, and the short-lived nuclear proteins these genes encode contain uniquely highly conserved domains (domains I–IV) [11]. Numerous studies have demonstrated that auxin-related genes regulate the formation of adventitious and lateral roots through interactions with auxin, such as IAA14, AXR3, IAA28, and IAA19 [12,13,14,15]. In rice, researchers have identified 24 members of the Aux/IAA family. The overexpression of OsIAA31 reduces adventitious root formation and confers insensitivity to auxin and gravitropic stimuli [15]. However, the mutation of the OsIAA11 gene severely inhibits lateral root primordium formation without affecting adventitious root development [16]. SlIAA15 plays a role in lateral root formation in tomatoes, and the density of root hairs decreases after inhibiting the expression of SlIAA15 [17]. In addition to participating in root development, the Aux/IAA gene family is also widely involved in non-biological stress in plants [18]. In rice, the overexpression of OsIAA6, which is induced by drought stress, increases drought tolerance by regulating growth hormone biosynthesis [19]. Meanwhile, silencing the OsIAA20 gene in rice significantly reduces the contents of proline and chlorophyll, leading to decreased drought and salt tolerance [20]. This indicates that Aux/IAA genes can enhance plant stress resilience.
Rosa hybrida is a perennial woody plant in a highly heterozygous group of hybrids formed by years of repeated complex crosses and long-term selection within and between species of the rose genus [21]. The characteristics of high heterozygosity provide a rich source of materials for the breeding and application of roses. However, they also make it difficult to study the physiological and molecular mechanisms of roses deeply. As a group with a very complex genetic background, different genotypes of roses have large differences in rooting ability, and there are significant differences in characteristics such as adventitious root occurrence frequency, the number of adventitious roots, and root length weight. In production practice, roses are usually obtained quickly with stable qualities by grafting and cutting to inherit the excellent characteristics of mother plants and significantly shorten the reproductive cycle. The rooting ability of the clones is a key factor determining the success of rose-cutting propagation. Therefore, elucidating the molecular mechanism underlying the rapid rooting of rose cuttings is crucial for improving the efficiency of rose propagation. Although most rose cuttings take root relatively easily, there are still many excellent clone cuttings that have difficulty taking root. At the same time, accomplishing genetic transformations in roses is in itself difficult, inefficient, and slow, which greatly limits in-depth research on the molecular mechanisms of roses. Therefore, clarifying the mechanism underlying the rapid rooting of rose cuttings at the genetic level has important theoretical significance and practical production value for improving the rapid reproduction efficiency of roses.
The Aux/IAA gene family is considered a key participant in auxin signal transduction and plays an important role in adventitious root formation. However, the molecular mechanism of the Aux/IAA gene family in the adventitious root formation of roses needs to be further studied. This study conducted a comprehensive analysis of the gene structure, conserved domains, motifs, and promoter cis-acting elements of the Aux/IAA gene family in roses using RNA-seq. It was found that the RhIAA25 gene plays a pivotal role in regulating adventitious root formation. This gene promotes the growth of primary roots in Arabidopsis and inhibits adventitious root development. The identification and preliminary functional analysis of the RhIAA25 gene offer crucial genetic resources for understanding the molecular mechanisms underlying adventitious root formation in roses.

2. Materials and Methods

2.1. Plant Materials

The experimental materials used in this study were R. hybrida “The Fairy”, Arabidopsis thaliana, Columbia, and Nicotiana benthamiana, which were planted in a mixture of vermiculite and peat (v/v = 1:1) and placed in an incubator at the Laboratory of Ornamental Plant Genetics and Breeding at Northeast Agricultural University (Harbin, China). The cultivation conditions were maintained at a photoperiod of 16 h of light and 8 h of darkness, having a temperature range of 22 ± 3 °C and relative humidity between 40 and 60%.

2.2. Identification of the Aux/IAA Gene Family in R. hybrida

To identify and analyze the RhAux/IAA gene family systematically, the genome sequence, protein sequence, and genome annotation files of Rosa chinensis were downloaded from the Genome database for Rosaceae (GDR) (https://www.rosaceae.org/, accessed on 11 February 2024). Furthermore, the protein-conserved domain of Aux/IAA (PF02309) was downloaded from the Pfam database. To identify the potential members of the RhAux/IAA genes, we used TBtools software (v 2.088) [22]. The ExPasy online resource (https://web.expasy.org/protparam, accessed on 27 February 2024) was employed for the comprehensive analysis of the physicochemical attributes of the RhAux/IAA proteins, encompassing parameters such as amino acid composition, molecular mass, isoelectric point, and instability indices. Furthermore, the Cell Ploc web-based platform (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/, accessed on 3 March 2024) was used to detect the subcellular localization of candidate genes and to forecast the subcellular compartments where these proteins are likely to be localized.

2.3. Analysis of the Phylogenetic Tree, Conserved Domain, Motif, and Gene Structure of RhAux/IAA

The ClustalW (https://www.genome.jp/tools-bin/clustalw, accessed on 3 March 2024) was employed to align the amino acid sequences obtained from A. thaliana and R. hybrida with parameters set to Gap Opening Penalty: 10 and Gap Extension Penalty: 0.2. Subsequently, the phylogenetic tree was constructed by using the maximum likelihood method with MEGA 11 software (https://www.megasoftware.net/, accessed on 22 February 2024) [23]. To enhance the stability of the tree, the Bootstrap parameter was set to 1000, and the rest of the parameters were kept as default. The RhAux/IAA amino acid sequences were submitted to NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 7 March 2024) to obtain the number and location information of the conserved domains using the Batch CD-Search tool. The conserved motifs of RhAux/IAA proteins were identified and analyzed by using the online tool MEME (https://meme-suite.org/meme/, accessed on 15 March 2024), for which the number of motifs was set to 10, the length of motifs was set to 6~100, and the rest parameters were kept as default values. Moreover, to identify the position of exons and introns of the RhAux/IAA genes, we used the GFF annotation file of the rose. Subsequently, TBtools software (v 2.088) was employed to visualize the phylogenetic tree, conserved domains, conserved motifs, and gene structure of RhAux/IAA.

2.4. Chromosomal Localization and Collinearity Analysis of RhAux/IAA

The GFF annotation file of the rose genome was downloaded from the GDR database, and the location information of RhAux/IAA genes on chromosomes was drawn and visualized by Gene Location Visualize from the GTF/GFF tool in TBtools software (v 2.088). The genome sequences and GFF annotation files of R. hybrida, Rosa rugosa, Fragaria vesca, and A. thaliana were downloaded from the GDR and Tair databases. The One Step MCScanX tool of TBtools software (v 2.088) was utilized to analyze and visualize gene duplication in R. hybrida and F. vesca, R. hybrida and R. rugosa, and R. hybrida and A. thaliana.

2.5. Analysis of Cis-Acting Elements in RhAux/IAA Genes

TBtools software (v 2.088) was used to extract the sequence 2000 bp upstream of the start codon of the RhAux/IAA genes as their promoter sequences based on the genome sequences and GFF annotation file. Following this, the extracted promoter sequences were uploaded to the PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 19 March 2024) for cis-acting elements analysis. The Basic Biosequence View tool in TBtools software (v 2.088) was used to visualize the cis-acting elements of RhAux/IAA promoter sequences.

2.6. Analysis of Expression of RhAux/IAA Genes during Rooting Based on RNA-Seq

One node cutting of R. hybrida “The Fairy” was cultivated in 1/2 Murashige and Skoog (MS) nutrient medium supplied with 2% sugar and 0.7% agar. The culture conditions were 16 h of light and 8 h of darkness, and the temperature was maintained at 22 ± 3 °C. The cuttings were sampled at 0, 1, 3, 5, 10, 15, and 20 days of post-treatment, with three biological replicates, and each biological replicate contained 10 different cuttings. Transcriptome sequencing was performed by Beijing Biomarker Technologies Co., Ltd. (Beijing, China). Gene expression data were obtained by cDNA library construction, high-throughput sequencing, and data analysis. Finally, the expression matrix of RhAux/IAA genes was extracted by TBtools software (v 2.088) and visualized.

2.7. Quantitative Real-Time PCR

Total RNA was extracted using the FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, China). Subsequently, the RNA was transcribed into cDNA using the Novazan Reverse Transcription Kit with HiScript II QRT SuperMix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China). The relative expression of the RhIAA25 gene was quantified with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) on the BIO-RAD CFX96 Touch system (CFX Touch; Bio-Rad, Hercules, CA, USA). The qRT-PCR thermal cycling conditions were as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s and 30 s at 60 °C. The 2−∆∆CT quantification method was used to calculate the relative expression levels. RhActin was used as an internal reference gene [24]. Gene primers were designed by using the GeneScript online tool (https://www.genscript.com/, accessed on 19 March 2024). The primers used in this study are mentioned in Table S1.

2.8. Subcellular Localization

The full-length RhIAA25 gene lacking a stop codon was inserted into BamHI and KpnI (Takara, Beijing, China) of the pCAMBIA1300-sGFP vector (Fenghui Biotechnology Co., Ltd., Changsha, China) by using the ClonExpress® II One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China). The constructed vector pGAMBIA1300-RhIAA25-sGFP was transformed into Agrobacterium tumefaciens GV3101, and subcellular localization was performed according to previous research [25]. Specifically, the infection solution 200 μmol acetosyringone (AS), 10 mmol/L 2-morpholinoethanesulphonic acid (MES), and 10 mmol/L MgCl2 containing A. tumefaciens GV3101 was injected into 4-week-old tobacco leaves using a syringe. After 2 days of incubation in the dark at 23 °C, the subcellular localization of RhIAA25 was visualized and photographed by using a laser-scanning confocal microscope at 488 nm (FV3000, Olympus, Shinjuku, Japan).

2.9. Yeast Self-Activation Analysis of RhIAA25

A pGBKT7-RhIAA25 vector was constructed by using the ClonExpress® II One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China). pGBKT7-53+pGADT7-T (positive control), pGBKT7-lam+pGADT7-T (negative control), and pGBKT7-RhIAA25+pGADT7 were transformed into Y2H gold yeast competent cells (WeiDi Biotechnology, Shanghai, China). Then, selected yeast colonies were transferred to SD/-Trp/-Leu liquid yeast medium and cultured at 28 °C at 4000 rpm for 1 min to collect the yeast cells. The Y2H gold yeast with plasmids was re-suspended in sterile water when the OD600 value reached 0.2. The suspended culture was diluted to 10×, 100×, and 1000×. The diluted bacterial solution was placed in SD/-Trp/-Leu/-Ade/-His solid medium and cultured at 28 °C. The self-activating activity of RhIAA25 was evaluated by yeast growth.

2.10. Genetic Transformation and Identification of Transgenic RhIAA25 in A. thaliana

The RhIAA25 open reading frame was amplified and cloned into the BamHI site of the pBI121 vector. The constructed pBI121-RhIAA25 was transformed into Agrobacterium tumefaciens GV3101 and cultured in YEP medium until the OD600 value became 0.5–1.0. The bacteria were collected after centrifugation at 5000 rpm for 5 min and then re-suspended in an infection solution (200 μmol AS, 10 mmol/L MES, and 10 mmol/L MgCl2). The genetic transformation of A. thaliana was performed by using an Agrobacterium-mediated method, as described in Tsuda et al. [26]. The DNA of transgenic A. thaliana was extracted by the CTAB method, and positive transgenic was identified by PCR. T3 generation transgenic Arabidopsis seeds were sterilized and sown in 1/2 Murashige and Skoog (MS) nutrient medium (30 g/L sucrose + 7 g/L agar powder). The root length phenotype of Arabidopsis primary roots was determined 7 days after planting. Meanwhile, the primary roots of the wild-type strain and three transgenic strains were removed after 7 days and transferred to 1/2 Murashige and Skoog (MS) nutrient medium (30 g/L sucrose + 7 g/L agar) to further observe the phenotypic changes in the adventitious roots. Each treatment consisted of three biological replicates, and each replicate contained ten plants. All treatments were carried out in an incubator with 16 h of light/8 h of dark at 22 °C.

2.11. Statistical Analyses

Statistical analyses were performed by using IBM SPSS v25.0 (SPSS Inc., Chicago, IL, USA). To compare the statistical validity of the data, the Least Significant Difference (LSD) test was performed. The significance level was set at p < 0.05. Three biological replicates were used for each assay. Furthermore, TBtools software (v 2.088) was used to create the conserved domains, motifs, gene structure, and heatmaps. Graphpad Prism 8.0.0 (GraphPad Software, San Diego, CA, USA) was used to plot graphs.

3. Results

3.1. Identification of RhAux/IAA Genes

Based on the comprehensive biosequence analysis employing profile hidden Markov models (HMMER) and the Basic Local Alignment Search Tool for proteins (BLASTP v2.16.0), coupled with the identification of the Aux/IAA (PF02309) domain utilizing Pfam, a total of 34 Aux/IAA members were successfully identified within the genomic dataset of R. hybirda (Table 1). Based on their chromosomal arrangement, these 34 genes were designated as RhIAA1-RhIAA34. Analysis of physicochemical properties revealed that the isoelectric point (pI) ranges from 5.00 (RhIAA22) to 9.65 (RhIAA19). The molecular weight (MW) of the predicted RhAux/IAA proteins varies from 11,445.23 Da (RhIAA21) to 130,207.21 Da (RhIAA5). The instability index (II) ranges between 20.32 (RhIAA21) and 69.93 (RhIAA5) (Table 1). In this study, we noted that with the exception of RhIAA12, RhIAA17, RhIAA21, RhIAA24, and RhIAA28, the majority of RhAux/IAA proteins exhibit instability (instability index > 40) [27]. The Aliphatic Index ranges from 59.10 (RhIAA26) to 89.49 (RhIAA21), whereas the Grand Average of Hydropathicity spans from −0.780 (RhIAA2) to −0.112 (RhIAA8). Furthermore, all the RhAux/IAA proteins are localized on the cell nucleus, aligning with the typical characteristics of transcription factors.

3.2. Phylogenetic Analysis of RhAux/IAA Genes

In order to investigate the relationship between Aux/IAA proteins in the rose (R. hybrida) and the model plant A. thaliana, a phylogenetic analysis was conducted by utilizing the complete amino acid sequences of predicted Aux/IAAs from both R. hybrida and A. thaliana. Each Aux/IAA protein was classified into three distinct clades, designated as Clade A, Clade B, and Clade C. Clade A comprised 28 members from A. thaliana, including AtIAA13, AtIAA26, AtIAA18, and AtIAA30, among others, and 22 members from rose, such as RhIAA13, RhIAA15, RhIAA26, etc. (Figure 1). Clade B consisted of only two members, AtIAA33 and RhIAA19 (Figure 1). Clade C comprised 11 rose members, including RhIAA14, RhIAA11, RhIAA23, RhIAA18, etc., with no homologous members from A. thaliana, indicating potential functional deviation in these members (Figure 1). Orthologous members typically exhibited similar biological functions, such as AtIAA16 and RhIAA12, AtIAA27, and RhIAA34 in Clade A, as well as RhIAA19 and AtIAA33 in Clade B.

3.3. Gene Structure Analysis of RhAux/IAA Family Members

Based on evolutionary relationship analysis, the RhAux/IAA gene family was categorized into the following three distinct clades: Clade A, Clade B, and Clade C (Figure 2a). All members of the RhAux/IAA gene family possessed the Aux-IAA superfamily domain. Notably, Clade C, in contrast to Clade A and Clade B, also harbored the Auxin-resp superfamily domain and the Bfil-C-EcoRII-N-B3 superfamily domain (Figure 2b). RhIAA18 stood out for its unique composition, encompassing not only these three domains but also the PABP-1234 superfamily domain (Figure 2b). The Multiple Em for Motif Elicitation (MEME) web server, encompassing 10 motifs, was employed to analyze the conserved motif distributions of RhAux/IAA proteins. Across these three clades, motif1, motif2, and motif7 were conserved in all members of the RhAux/IAAs. However, Clade C exhibited a greater diversity of motifs, with all nine motifs present (Figure 2c). These findings implied that, with few exceptions of specialized proteins, members within the same branch of the RhAux/IAAs exhibited similarity in motif position and quantity, suggesting potential functional conservation. To further explore the structural attributes of RhAux/IAA genes, an analysis of their exon–intron gene structures was conducted. The intron counts in RhAux/IAA genes varied from 1 to 14, with Clade A exhibiting a lower number, predominantly ranging from 1 to 7. Conversely, Clade C featured a higher intron count, mainly distributed in the range of 12 to 14, whereas Clade B was characterized by the presence of a single intron (Figure 2d). These observations suggested that genes within the same evolutionary branch tend to exhibit analogous intron number distributions.

3.4. The Chromosomal Localization and Collinearity Analysis of the RhAux/IAA Genes

The RhAux/IAA genes were distributed across seven chromosomes, with the densest distribution on chromosome 2, which harbored nine RhAux/IAA genes (Figure 3a). Following this, chromosome 4 contained eight RhAux/IAA genes, while six genes were found to be present on chromosomes 6 and 7. Chromosome 3 contained three RhAux/IAA genes, and chromosomes 1 and 5 contained only one gene (Figure 3a). Additionally, eight RhAux/IAA genes were identified as tandem duplicates, located on chromosomes 2, 3, 4, and 6 (Figure 3a). Previous studies also indicated that tandem duplications contribute to the expansion of this gene family. Gene collinearity analysis can elucidate the evolutionary relationships among the genomes of different species or individuals. Examining the collinearity among genes facilitates the investigation of the origin, evolution, and functional diversity of gene families. To further explore the phylogenetic mechanism of the RhAux/IAA genes and to identify the evolutionary relationships of the RhAux/IAAs among different species, we conducted collinearity analysis between R. hybrida and Fragaria vesca, R. rugosa, and A. thaliana. The results revealed the presence of 46 homologous gene pairs between R. hybrida and F. vesca, 41 between R. hybrida and R. rugosa, and 28 between R. hybrida and A. thaliana (Figure 3b).

3.5. Cis-Elements in the Promoters of RhAux/IAA Genes

Cis-acting elements located within gene promoters are involved in modulating gene expression in response to biotic and abiotic stresses, achieved through their capacity to bind to transcription factors. This regulatory mechanism profoundly influences the growth and development of organisms. To examine the potential regulatory functions of RhAux/IAAs in plant growth, development, and stress responses, we conducted an in-depth analysis of the cis-regulatory elements present in their promoter regions. The obtained results revealed that these elements in the RhAux/IAA genes can be categorized into four primary types as follows: hormone-related, light-responsive, development-related, and abiotic stress-related. Notably, we identified elements associated with growth and development (G-Box, MBS, circadian), abscisic acid responsiveness, gibberellin responsiveness, auxin responsiveness, MeJA responsiveness, salicylic acid responsiveness (TGA-element, ABRE, CGTCA-motif, TATC-box, TGACG-motif, P-box, TCA-element), stress responsiveness (CAT-box, ARE, GCN4_motif, LTR, A-box), and light responsiveness (GATA-motif, TCCC-motif, MRE, AE-box, ACE, Sp1, among others) (Figure 4). These findings suggested that RhAux/IAA genes may be crucial in mediating responses to hormonal signals and abiotic stresses.

3.6. The Expression Pattern of RhAux/IAA Genes during Adventitious Root Development

The Aux/IAA gene family is extensively recognized to play a key role in the morphogenesis of plant roots. To delineate the potential functions of RhIAAs in the emergence of adventitious roots in roses, we conducted an analysis of the transcriptomic dataset of R. hybrida “The Fairy” stem segments across various time intervals spanning from self-cutting to the pre-adventitious root formation phase. This dataset was procured from our laboratory’s preceding research endeavors (Table S2). Our findings identified that all 34 RhAux/IAA genes were expressed in a typical manner within the stem segments (Figure 5). Notably, the expression levels of RhIAA19, 23, 2, 8, 7, and 25 were observed to be relatively diminished during the 0–3-day period, while the remaining 28 genes exhibited relatively elevated expression levels on day 0 (Figure 5). Furthermore, a significant surge in the expression levels of RhIAA10, 27, and 29 was noted after three days of treatment, implying their potential involvement in the induction and initiation stages of adventitious root formation (Figure 5). Additionally, a marked increase in the expression levels of RhIAA7 and RhIAA25 was observed from days 5 to 25 subsequent to treatment, suggesting their vital roles in fostering adventitious root proliferation (Figure 5). Previous research has elucidated the significant roles of AtIAA7 and AtIAA14 in modulating the development of adventitious roots in Arabidopsis thaliana. In our investigation, RhIAA25 demonstrated the closest affinity with AtIAA7 [28] and AtIAA14 [29] and was prominently expressed during the generation and maturation stage of adventitious roots in roses. Collectively, these results identified the functional significance of RhIAA25 in the regulation of adventitious root emergence.

3.7. The Expression Pattern and Molecular Characteristics of RhIAA25

The expression patterns of the RhIAA25 gene in different rose organs were analyzed by qRT-PCR. The results revealed that the RhIAA25 gene exhibited the highest expression level in rose stems, approximately 9-fold higher than in roots and stamens (Figure 6a). Most adventitious roots are mainly derived from stems [4]. This suggests that RhIAA25 may play an essential role in adventitious root formation in roses. Confocal laser scanning microscopy was employed to visualize fluorescence signals. The green fluorescence signal of the pCAMBIA1300-RhIAA25-sGFP vector was exclusively observed in the nucleus, whereas in the control group, the fluorescence of pCAMBIA1300-sGFP leaves was diffusely distributed and visible in both the nucleus and the cell membrane, indicating that RhIAA25 is localized on the nucleus (Figure 6b). Subsequently, we assessed the transcriptional activity of RhIAA25. The experimental result demonstrated that Y2H gold yeast colonies transformed with the pGBKT7-53/pGADT7-T plasmid exhibited normal growth on SD/-Trp/-His/-Leu/-Ade plates, whereas the experimental group transformed with the pGBKT7-RhIAA25/pGADT7 plasmid did not grow normally, aligning with the negative control transformed with the pGBKT7-lam/pGADT7-T plasmid (Figure 6c). These results suggested that RhIAA25 lacks transcriptional self-activation activity.

3.8. The Effect of Overexpressing RhIAA25 on Arabidopsis Root Development

To examine the impact of RhIAA25 on root growth, we generated transgenic A. thaliana lines with RhIAA25 overexpression via Agrobacterium-mediated genetic transformation. We selected three Arabidopsis transgenic lines exhibiting high RhIAA25 transcription levels (#12, #13, and #20) for the analysis of primary and adventitious root development (Figures S1 and S2). Cultivation on 1/2 (MS) nutrient medium revealed that RhIAA25 overexpression significantly enhanced primary root elongation and accelerated the growth rate compared with the wild-type (Figure 7a). The transgenic lines displayed an average root length of approximately 2.5 cm, whereas the wild-type had a root length of 1.7 cm (Figure 7b). To investigate the influence of the RhIAA25 gene on adventitious root formation in Arabidopsis, we cultured the seeds for 7 days on a hormone-free medium. Subsequently, under sterile conditions, we excised the primary roots and replanted them on the medium for an additional 10 days to monitor adventitious root development. Phenotypic analysis indicated that the wild-type lines exhibited superior growth and longer adventitious roots compared with the transgenic lines (Figure 7c), with the adventitious root length of the wild-type lines being approximately 1.5 cm, which was significantly longer than that of the transgenic lines, whereas the transgenic lines displayed an average root length of approximately 0.5 cm (Figure 7d). These findings suggest that RhIAA25 is a vital regulator of plant root development, facilitating primary root elongation during the early stages of Arabidopsis growth while inhibiting adventitious root formation.

4. Discussion

The Aux/IAA gene family plays an important role in plant growth and development [30,31]. In this study, 34 members of the Aux/IAA gene family were identified, which were distributed on seven chromosomes of the rose. In the model plant (A. thaliana), the function of the Aux/IAA gene family has been studied deeply [32]. In order to understand the possible function of Aux/IAA genes in roses, we constructed the phylogenetic tree of Aux/IAA in Arabidopsis and the rose. According to the evolutionary relationship, they were divided into Clade A, Clade B, and Clade C. Most of the Aux/IAA family members in the rose and Arabidopsis were assigned to Clade A, but Clade C only contained Aux/IAA gene members in the rose. At the same time, the data of the conserved domain, motif, and gene structure analysis showed that Clade C contained more domains, motifs, and exons than Clade A and Clade B. These data suggested that the function of Aux/IAA in Clade C may be different from that in Clade A and Clade B. These results are slightly different from those of Panax ginseng, Malus pumila, and Raphanus sativus [18,33,34], which may be due to the different functions of Aux/IAA in different plants. These analyses help to infer the function of Aux/IAA in roses according to the known function of Aux/IAA in Arabidopsis and lay a foundation for the subsequent mining of related genes.
Cis-acting elements play an important role in the regulation of gene expression in plants. Analyzing cis-acting elements can help determine the possible functions of genes. Through yeast heterologous expression and overexpression in Arabidopsis, the DgIAA21 gene was identified as a key gene for enhancing drought resistance in Dactylis glomerata [35]. Additionally, overexpression of the MdIAA9 gene enhanced proline content in transgenic tobacco and reduced MDA levels, thus improving drought resistance in tobacco [36].
To investigate the potential cis-acting elements in the promoters of RhAux/IAA genes, an analysis was conducted, revealing a rich composition of regulatory elements. These include growth and development-related motifs such as G-box, MBS, and circadian elements; stress response elements like CAT-box, ARE, GCN4_motif, LTR, and A-box; and light response elements comprising GATA-motif, TCCC-motif, MRE, AE-box, ACE, and Sp1. These results indicated that RhAux/IAA may play an important role in growth, development, and stress responses. Aux/IAA is an early responsive gene to auxin; through the analysis of its promoter cis-acting elements, we also found that the promoter contains a large number of hormone response elements, including auxin, gibberellin, and abscisic acid response elements. These results further suggested that these hormones may be involved in the growth and development of roses by regulating the expression of Aux/IAA genes.
The Aux/IAA gene family plays an important role in the process of adventitious root development in plants [37,38,39]. In A. thaliana, AtIAA14 was demonstrated to regulate the development of lateral roots [29]. Meanwhile, OsIAA9 facilitates the proliferation of lateral roots and is involved in the abrogation of the gravitropic response and the depletion of starch granules in root apices [40]. By analyzing the expression patterns of RhAux/IAAs, it was noted that RhAux/IAAs were upregulated between 5 and 20 days post-cutting, indicating that RhAux/IAAs may play a vital role in the later regulation of rose adventitious roots (Figure 5). The AtIAA7 and AtIAA14 genes play important roles in lateral and adventitious root development [25,26]. The results of the Aux/IAA phylogenetic tree and expression pattern analysis indicated that RhIAA25 is a homologous gene of AtIAA7 and AtIAA14, and its expression was significantly upregulated during the rooting process, suggesting that this gene may play an important role in the development of adventitious roots in roses. To further investigate the role of RhIAA25 in roses, we conducted subcellular localization and transcriptional self-activation activity analysis, and it was noted that RhIAA25 encoded a nucleus protein (Figure 6b) [41]. The transcriptional activation assay revealed that RhIAA25 lacks transcriptional self-activation activity, implying its potential utility for future screening of interacting genes (Figure 6c). Furthermore, transgenic experiments revealed that the overexpression of RhIAA25 suppresses the formation of adventitious roots to a certain extent but promotes the growth of primary roots (Figure 7). These results aligned with previous findings on the overexpression of AtIAA6, AtIAA9, and AtIAA17 in Arabidopsis, which also resulted in the suppression of adventitious root formation [28]. However, the results differed from the overexpression of TaIAA8. The overexpression of TaIAA8 inhibited adventitious root formation in transgenic plants, with little impact on primary root growth, indicating that TaIAA8 is mainly involved in the growth and development of lateral roots in plants. In R. hybrida “The Fairy”, the overexpression of RhIAA25 not only suppressed adventitious root formation but also promoted primary root growth in transgenic plants. These results further suggested that the AUX/IAA gene family plays a highly complex role in regulating root development [40,42]. The accumulating evidence also suggested that the Aux/IAA gene family plays an equally important role in stem elongation and leaf expansion [43]. Hence, the ongoing validation of the biological functions of Aux/IAA genes in roses and the exploration of their potential involvement in the regulation of homeostasis and signaling transduction of other hormones will be the primary focus of our future research.

5. Conclusions

In this study, a total of 34 RhAux/IAA gene family members were identified. Phylogenetic relationships divided the RhAux/IAA gene family into three clades, including Clade A, Clade B, and Clade C. The chromosome mapping results showed that the RhAux/IAA gene family was distributed on seven chromosomes of the rose, with the largest distribution on chromosome 2, having a total of nine genes. The cis-acting elements of the promoter indicated that the promoters of this gene family contain a large number of growth-related elements and light-responsive elements. In addition, through transcriptome analysis, the differentially expressed gene RhIAA25 was screened during adventitious root formation. RhIAA25 was subcellularly localized, self-activated in yeast, and expressed specifically. The results indicated that RhIAA25 was localized on the nucleus of the cell, with no yeast self-activation activity, and the expression level in the stem was found to be significantly increased. Subsequently, the model plant Arabidopsis was overexpressed, and the results revealed that the overexpression of RhIAA25 promoted the elongation of Arabidopsis’s main roots. On the contrary, the overexpression of RhIAA25 inhibited the development and growth of adventitious roots. This research provides a new insight into the functional role of the RhAux/IAA gene family in the process of adventitious root formation. These findings will help to further study the mechanism of adventitious root formation during the rapid propagation of roses and provide an important theoretical basis for the cutting propagation of roses.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14092005/s1. Figure S1: Detection of transgenic Arabidopsis thaliana using agarose gel electrophoresis; Figure S2: Determination of transcription levels in Arabidopsis overexpressing RhIAA25; Table S1: Primer information table; Table S2: FPKM value of RhAux/IAA genes.

Author Contributions

W.Z.: investigation, formal analysis, writing—original draft, and software. Y.Z.: data curation and formal analysis. M.H.: data curation and visualization. Y.F.: data curation and software. J.Z.: software and investigation. T.Y.: visualization and software. D.C.: visualization and data curation. J.D.: funding acquisition, supervision, methodology, and resources. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the China Postdoctoral Science Foundation (2023M730535).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We appreciate all the people who collaborated on this project.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic relationship of Aux/IAA family genes in R. hybrida. and A. thaliana. Proteins from R. hybirda and A. thaliana are denoted by Rh and At prefixes, respectively. Three major phylogenetic groups designated from Clade A to Clade C are indicated. The three clades are indicated by different colors. Blue circles represent A. thaliana and red stars represent R. hybrida.
Figure 1. Phylogenetic relationship of Aux/IAA family genes in R. hybrida. and A. thaliana. Proteins from R. hybirda and A. thaliana are denoted by Rh and At prefixes, respectively. Three major phylogenetic groups designated from Clade A to Clade C are indicated. The three clades are indicated by different colors. Blue circles represent A. thaliana and red stars represent R. hybrida.
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Figure 2. Phylogenetic relationships, conserved structural domains, and conserved motifs of RhAux/IAAs in R. hybrida. (a) Phylogenetic analysis. (b) Motif composition of RhAux/IAAs proteins, with different colors representing ten distinct motifs. (c) Conserved structural domains of RhAux/IAAs, with different colors indicating different structural domains. (d) Light green rectangles specify untranslated regions (UTRs); yellow rectangles represent exons; and black lines indicate introns.
Figure 2. Phylogenetic relationships, conserved structural domains, and conserved motifs of RhAux/IAAs in R. hybrida. (a) Phylogenetic analysis. (b) Motif composition of RhAux/IAAs proteins, with different colors representing ten distinct motifs. (c) Conserved structural domains of RhAux/IAAs, with different colors indicating different structural domains. (d) Light green rectangles specify untranslated regions (UTRs); yellow rectangles represent exons; and black lines indicate introns.
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Figure 3. Chromosomal localization and synteny analysis of RhAux/IAA genes. (a) Localization of RhAux/IAAs on all seven chromosomes of R. hybirda, Chromosomal location and synteny analysis of RhAux/IAAs. (b) Comparative analysis of the RhAux/IAA gene family among R. hybrida, F. vesca, R. rugosa, and A. thaliana. Red lines represent collinear RhAux/IAA gene pairs.
Figure 3. Chromosomal localization and synteny analysis of RhAux/IAA genes. (a) Localization of RhAux/IAAs on all seven chromosomes of R. hybirda, Chromosomal location and synteny analysis of RhAux/IAAs. (b) Comparative analysis of the RhAux/IAA gene family among R. hybrida, F. vesca, R. rugosa, and A. thaliana. Red lines represent collinear RhAux/IAA gene pairs.
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Figure 4. Diversity of cis-regulatory elements within RhAux/IAA promoter regions, represented by distinct colors.
Figure 4. Diversity of cis-regulatory elements within RhAux/IAA promoter regions, represented by distinct colors.
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Figure 5. Heatmap showing expression levels (FPKM) of RhAux/IAA family genes during rose adventitious rooting.
Figure 5. Heatmap showing expression levels (FPKM) of RhAux/IAA family genes during rose adventitious rooting.
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Figure 6. RhIAA25 tissue-specific expression, subcellular localization, and transcriptional activity. (a) Tissue-specific expression of RhIAA25. (b) Subcellular localization of RhIAA25 in tobacco leaves. pCAMBIA1300-sGFP empty vector was used as a control. (c) The growth of yeast cells co-transformed with various vectors on synthetic glucose SD/-Trp-His medium. pGADT7+pGBKT7-53 served as the positive control, while pGADT7+pGBKT7-LaminC was utilized as the negative control. The yeast cultures were diluted to 1×, 10×, 100×, and 1000×, corresponding to the original concentration (OD600 = 0.5), and 10-fold, 100-fold, and 1000-fold dilutions, respectively. Different letters indicate significant differences (p < 0.05).
Figure 6. RhIAA25 tissue-specific expression, subcellular localization, and transcriptional activity. (a) Tissue-specific expression of RhIAA25. (b) Subcellular localization of RhIAA25 in tobacco leaves. pCAMBIA1300-sGFP empty vector was used as a control. (c) The growth of yeast cells co-transformed with various vectors on synthetic glucose SD/-Trp-His medium. pGADT7+pGBKT7-53 served as the positive control, while pGADT7+pGBKT7-LaminC was utilized as the negative control. The yeast cultures were diluted to 1×, 10×, 100×, and 1000×, corresponding to the original concentration (OD600 = 0.5), and 10-fold, 100-fold, and 1000-fold dilutions, respectively. Different letters indicate significant differences (p < 0.05).
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Figure 7. Phenotypic assessment and length measurement of primary and adventitious roots in Arabidopsis overexpressing RhIAA25. (a) Phenotypic observation of Arabidopsis primary roots 7 days post-germination. (b) The length of the primary roots of wild-type and RhIAA25-overexpression lines. (c) Excision of primary roots from one-week-old wild-type and RhIAA25-overexpression Arabidopsis seedlings, followed by growth on Murashige and Skoog (MS) nutrient medium for ten days. Phenotypic observation of adventitious roots in seedlings without primary roots. (d) The length of adventitious roots of wild-type and RhIAA25-overexpression lines. Different letters indicate significant differences (p < 0.05).
Figure 7. Phenotypic assessment and length measurement of primary and adventitious roots in Arabidopsis overexpressing RhIAA25. (a) Phenotypic observation of Arabidopsis primary roots 7 days post-germination. (b) The length of the primary roots of wild-type and RhIAA25-overexpression lines. (c) Excision of primary roots from one-week-old wild-type and RhIAA25-overexpression Arabidopsis seedlings, followed by growth on Murashige and Skoog (MS) nutrient medium for ten days. Phenotypic observation of adventitious roots in seedlings without primary roots. (d) The length of adventitious roots of wild-type and RhIAA25-overexpression lines. Different letters indicate significant differences (p < 0.05).
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Table 1. Physical and chemical characteristics of RhAux/IAA proteins in R. hybrida.
Table 1. Physical and chemical characteristics of RhAux/IAA proteins in R. hybrida.
Gene NameGene IDIsoelectric Point (pl)Molecular Weight (Da)Instability IndexAliphatic IndexGrand Average of
Hydropathicity		Subcellular Localization
RhIAA1Chr1g03600416.5776,056.9652.0270.87−0.444Nucleus
RhIAA2Chr2g00877918.4024,832.9747.9470.51−0.780Nucleus
RhIAA3Chr2g00898018.7136,549.6340.3663.03−0.678Nucleus
RhIAA4Chr2g00912617.5933,159.6645.3269.00−0.427Nucleus
RhIAA5Chr2g00955516,22130,207.2169,9369.70−0.707Nucleus
RhIAA6Chr2g01098818.0940,175.0748.9461.08−0.601Nucleus
RhIAA7Chr2g01373016.6021,507.4658.1971.95−0.457Nucleus
RhIAA8Chr2g01373115.1512,409.5246.6578.91−0.112Nucleus
RhIAA9Chr2g01393018.0937,084.9247.9262.90−0.615Nucleus
RhIAA10Chr2g01406819.1636,783.4252.4368.39−0.699Nucleus
RhIAA11Chr2g01529516.00101,532.4766.7772.86−0.490Nucleus
RhIAA12Chr3g04510716.0527,871.5133.9967.83−0.596Nucleus
RhIAA13Chr3g04510818.2420,409.4345.6260.88−0.646Nucleus
RhIAA14Chr3g04877715.7994,404.8366.8369.33−0.509Nucleus
RhIAA15Chr4g03896116.4121,983.1241.4375.98−0.531Nucleus
RhIAA16Chr4g03896218.5926,427.6142.1767.47−0.470Nucleus
RhIAA17Chr4g03955118.1839,694.8638.5572.41−0.301Nucleus
RhIAA18Chr4g03977716.24124,586.5864.8570.86−0.598Nucleus
RhIAA19Chr4g04024419.6518,232.6765.4586.91−0.478Nucleus
RhIAA20Chr4g04280117.1923,209.9450.7279.38−0.505Nucleus
RhIAA21Chr4g04287915.7611,445.2320.3289.49−0.249Nucleus
RhIAA22Chr4g04343915.0021,066.5549.2281.08−0.481Nucleus
RhIAA23Chr5g00149616.0498,764.0965.7873.67−0.434Nucleus
RhIAA24Chr6g02866016.3432,064.5939.6666.08−0.394Nucleus
RhIAA25Chr6g02895716.4626,908.6942.5762.08−0.629Nucleus
RhIAA26Chr6g02895815.2221,129.7449.0859.10−0.748Nucleus
RhIAA27Chr6g02925515.8375,288.0160.2870.36−0.411Nucleus
RhIAA28Chr6g02978215.9326,495.8037.5872.01−0.712Nucleus
RhIAA29Chr6g03025515.22100,600.1457.2774.22−0.391Nucleus
RhIAA30Chr6g03066318.2134,123.5154.4570.59−0.721Nucleus
RhIAA31Chr7g01860816.5274,700.3051.1572.81−0.482Nucleus
RhIAA32Chr7g01889116.1593,554.8153.6065.06−0.656Nucleus
RhIAA33Chr7g02197716.0375,041.5360.2570.89−0.488Nucleus
RhIAA34Chr7g02331017.0539,188.3947.8971.21−0.578Nucleus
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Zhang, W.; Zhang, Y.; Huangfu, M.; Fan, Y.; Zhang, J.; Yang, T.; Che, D.; Dong, J. Genome-Wide Identification and Analysis of the Aux/IAA Gene Family in Rosa hybrida—“The Fairy”: Evidence for the Role of RhIAA25 in Adventitious Root Development. Agronomy 2024, 14, 2005. https://doi.org/10.3390/agronomy14092005

AMA Style

Zhang W, Zhang Y, Huangfu M, Fan Y, Zhang J, Yang T, Che D, Dong J. Genome-Wide Identification and Analysis of the Aux/IAA Gene Family in Rosa hybrida—“The Fairy”: Evidence for the Role of RhIAA25 in Adventitious Root Development. Agronomy. 2024; 14(9):2005. https://doi.org/10.3390/agronomy14092005

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Zhang, Wuhua, Yifei Zhang, Minge Huangfu, Yingdong Fan, Jinzhu Zhang, Tao Yang, Daidi Che, and Jie Dong. 2024. "Genome-Wide Identification and Analysis of the Aux/IAA Gene Family in Rosa hybrida—“The Fairy”: Evidence for the Role of RhIAA25 in Adventitious Root Development" Agronomy 14, no. 9: 2005. https://doi.org/10.3390/agronomy14092005

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