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Article

Characterization of OfERF17 as a Key Regulator of Petal Senescence in Osmanthus fragrans

by
Gongwei Chen
1,2,†,
Dandan Zhang
1,†,
Fengyuan Chen
1,
Yixiao Zhou
1,
Heng Gu
1,
Xuyang Qin
1,
Yuanzheng Yue
1,
Lianggui Wang
1,* and
Xiulian Yang
1,*
1
Key Laboratory of Landscape Architecture, College of Landscape Architecture, Nanjing Forestry University, No. 159 Longpan Road, Nanjing 210037, China
2
School of Landscape Architecture, Jiangsu Vocational College of Agriculture and Forestry, 19 Wenchang East Road, Jurong 212400, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2025, 16(4), 615; https://doi.org/10.3390/f16040615
Submission received: 22 February 2025 / Revised: 10 March 2025 / Accepted: 27 March 2025 / Published: 31 March 2025
(This article belongs to the Section Genetics and Molecular Biology)
Browse Figures
Versions Notes

Abstract

:
Osmanthus fragrans is a highly valued ornamental tree species in China, but its short flowering period limits its ornamental appeal. Investigating the mechanisms of flower senescence in O. fragrans is therefore of significant importance. Ethylene, a key endogenous hormone, plays a central role in flower senescence, and the AP2/ERF gene family, which includes ethylene response factors, is known to regulate this process in various plants. Transcriptome sequencing and expression analysis identified OfERF17 as a critical gene influencing petal senescence in O. fragrans. Bioinformatics analysis revealed that OfERF17 lacks transmembrane transport structures but contains multiple phosphorylation sites and shares a close phylogenetic relationship with the Olea europaea var. Sylvestris. Subcellular targeting and yeast-based auto-activation tests revealed that OfERF17 resides in the nucleus and possesses a transcriptional self-activation capability. Transient expression studies conducted in O. fragrans petals indicated a decrease in the expressions of two genes associated with senescence, namely, OfSAG21 and OfACO3, when compared to the control group. Additionally, the levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA) were markedly reduced. Transgenic Nicotiana tabacum blooms one day more than the wild type, and NtSAG12 and NtACO1 expressions were lower than wild type. These results suggest that OfERF17 functions to delay petal senescence in O. fragrans. This study enhances our knowledge of the molecular mechanisms underlying O. fragrans petal senescence and provides insights into strategies for prolonging its flowering period.

1. Introduction

Osmanthus fragrans, a member of the Osmanthus genus in the Oleaceae family, is an evergreen shrub or tree renowned as one of China’s top ten traditional famous flowers. It is highly valued not only as an ornamental species, but also as a fragrant plant with significant cultural, ecological, and medicinal importance, making it widely utilized in horticulture [1]. The dense foliage of O. fragrans remains vibrant throughout the year, and the plant can bloom even at the seedling stage, exhibiting a prolonged flowering period and a strong, pleasant fragrance. These characteristics contribute to its substantial economic and developmental potential [2]. However, O. fragrans exhibits pronounced aging symptoms within 4–5 days after flowering, resulting in a short ornamental flowering period. This senescence process irreversibly diminishes its ornamental and commercial value [3]. Consequently, extending the flowering period of O. fragrans has emerged as a critical challenge. Despite its significance, research on the senescence mechanisms of O. fragrans petals remains limited in China, underscoring the importance of investigating this phenomenon to enhance its ornamental and economic value.
Petal senescence is a highly regulated form of programmed cell death that occurs following tissue differentiation and petal maturation [4]. This process involves significant cytological, physiological, and molecular changes, driven by complex interactions among various plant growth regulators [5]. While senescence progresses at varying rates across different floral tissues, these components remain interconnected [6]. Extensive research highlights ethylene as a critical hormone regulating both flowering and senescence in plants [7,8,9]. Certain osmanthus varieties exhibit ethylene sensitivity, with endogenous ethylene production—independent of pollination and ovary development—serving as a key regulator of flower senescence [10,11]. Current molecular studies primarily focus on ethylene biosynthesis and signaling pathways to elucidate mechanisms for extending the flowering period and regulating flower development. These efforts aim to selectively inhibit ethylene biosynthesis and signaling to delay flower aging [12,13,14]. Nevertheless, the molecular processes governing the transition from flowering to senescence remain poorly understood.
The ethylene signaling pathway in the model plant Arabidopsis thaliana is well-established, following the sequence ethylene → ETR family receptors → CTR family proteins → EIN2 → EIN3/EIL transcription factors → ERF proteins → expression of ethylene-responsive genes [15]. Across different plant species, key genes involved in ethylene biosynthesis and signaling have been discovered. Ethylene response factors (ERFs), downstream elements of the ethylene signaling pathway, are members of the AP2/ERF transcription factor superfamily that is plant-specific and widely distributed [16]. The participation of ERF genes in the ripening of fruits has been reported in Solanum lycopersicum [17], Actinidia chinensis [18], and Malus pumila [19], while their role in abiotic stress responses has been explored in A. thaliana [20] and Populus [21]. In Rosa rugosa [22] and S. lycopersicum [23], silencing ERF genes results in either accelerated or delayed petal senescence. These studies collectively highlight the significant role of ERF genes in petal senescence.
In the initial phase of our research, we analyzed the AP2/ERF gene family in osmanthus flowers and identified 298 family members, including 247 ERF transcription factors. From these, 20 genes with high expression levels and significant differential expression were selected for qRT-PCR validation. The results demonstrate that 17 genes were highly expressed in the terminal flowering phase, 2 genes in the peak flowering phase, and 1 gene in both the bud—eye phase and the early flowering phase [24]. However, the functional roles of the ERF genes in osmanthus flower senescence remain poorly understood and require further investigation. Transcriptomic data reveal that OfERF17 is highly expressed during late flowering stages, suggesting its potential involvement in flower senescence, although its specific function remains unclear. To address this, we conducted a comprehensive bioinformatics analysis of OfERF17 and performed functional validation through transient overexpression experiments in osmanthus petals and transgenic Nicotiana tabacum. Phenotypic changes and gene expression trends were analyzed to elucidate the role of OfERF17. This study aims to provide new insights into the transcriptional regulation of floral senescence and establish a theoretical foundation for extending flowering time.

2. Materials and Methods

2.1. Plant Materials

The study utilized floral samples from O. fragrans ‘Rixiang Gui’ plants, aged 8–10 years, cultivated robustly at Nanjing Forestry University, encompassing five distinct developmental stages (the bud–pedicel phase, bud–eye phase, early flowering phase, peak flowering phase, and terminal flowering phase). For subcellular localization, 30-day-old seedlings of Nicotiana benthamiana were used, and Agrobacterium-mediated transformation was performed on 60-day-old wild-type N. tabacum ‘K326’. N. benthamiana and N. tabacum ‘K326’ were grown under controlled conditions: a 16 h light/8 h dark photoperiod, light intensity of 150 μmol/(m2·s), temperature of 23 °C, and relative humidity ranging from 50% to 80%.

2.2. RNA Extraction and Expression Analysis

Based on transcriptomic data from our research group (SRA: PRJNA932144), we analyzed the AP2/ERF gene family in O. fragrans and identified an ARF/ERF transcription factor, OfERF17. OfERF17 exhibited high expression levels and significant differences across five flowering stages, suggesting its potential involvement in O. fragrans flower senescence [24]. Consequently, OfERF17 was selected for functional validation. RNA was isolated from five distinct flowering stages of O. fragrans employing the TRNzol Universal Kit (Tiangen, Beijing, China) (Figure 1). Subsequently, cDNA synthesis was carried out using a reverse transcription kit (Tianroot, Beijing, China), which then acted as the template for qRT-PCR analysis. The OfRNA gene serves as a reference gene for normalizing the expression patterns of O. fragrans genes [25]. Primer Premier 5 software was utilized to design specific primers targeting OfERF17 (see Supplementary Materials Table S1). For each sample, Quantitative Real-Time PCR (qRT-PCR) was conducted in triplicate, encompassing both biological and technical replicates. The Qubit® 3.0 fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA) and SYBRGreen Pro Taq HS premixed qPCR kits (ICory, Changsha, China) were employed to perform the quantitative real-time PCR assays.

2.3. Bioinformatics Analysis of OfERF17 Genes

Initially, the Expasy ProtParam 3.0 tool was utilized to analyze the physicochemical characteristics of the protein encoded by OfERF17 (https://web.expasy.org/protparam/, accessed on 2 August 2023) [26]. Furthermore, the conserved domains of OfERF17 were identified through the NCBI website (https://www.ncbi.nlm.nih.gov/, accessed on 9 September 2023). The online software GOR IV was employed to predict the secondary structure of the OfERF17 protein (https://npsa.lyon.inserm.fr//cgi-bin/npsa_automat.pl?page=/NPSA/npsa_gor4.html, accessed on 9 September 2023) [27]. Hydrophilicity and hydrophobicity predictions were performed using the ExPASy Protscale 3.0 tool (https://web.expasy.org/protscale/, accessed on 26 August 2023). The transmembrane structure of OfERF17 was analyzed using the TMHMM-2.0 server (https://services.healthtech.dtu.dk/services/TMHMM-2.0/, accessed on 9 September 2023). Phosphorylation site prediction was carried out using the NetPhos-3.1 server (https://services.healthtech.dtu.dk//services/NetPhos-3.1/, accessed on 9 September 2023). The amino acid sequence of OfERF17 was compared using the BLAST 2.11.0 tool available on the NCBI website (https://www.ncbi.nlm.nih.gov/, accessed on 9 September 2023). Amino acid sequences homologous to OfERF17 from various species were retrieved, and a phylogenetic tree was generated utilizing MEGA 11.0 software. Additionally, multiple sequence alignment of the OfERF17 sequences was carried out with DNAMAN 9.0 software.

2.4. Cloning and Characterization of the Gene Sequence of OfERF17

Based on gene expression analysis and the mass status of the samples, cDNA from the S4 stage was selected as the template. First, the restriction sites of the OfERF17 sequence were identified using BioXM 2.6 software. The pSuper1300 vector was then digested with HindIII and KpnI endonucleases. Specific primers were designed using CE Design V1.04 (Supplementary Materials Table S1) for PCR amplification. Bands of the correct length were excised and purified. The digested vector was ligated with the target gene and transformed into E. coli DH5α. Ten single colonies were randomly selected for positive PCR screening, and colonies with the correct band length were submitted to Qingke Biotechnology Company (Qingke, Nanjing, China) for sequencing. Recombinant plasmids from sequence-verified E. coli were transformed into Agrobacterium GV3101 (Supplementary Materials Figure S1). PCR-positive colonies with the correct band length were confirmed, and the bacterial suspension was preserved at −80 °C for future applications.

2.5. Subcellular Localization and Transcriptional Activation Activity of the OfERF17 Proteins

Initially, the subcellular localization of OfERF17 was forecasted using the WoLF PSORT online tool (https://wolfpsort.hgc.jp/, accessed on 17 September 2023). The fusion vector pSuper1300-OfERF17, constructed as described above, contains a GFP signal for subcellular localization experiments. The marker plasmid was transformed into Agrobacterium tumefaciens, and the constructed Agrobacteria containing pSuper1300-OfERF17-GFP and pSuper1300-GFP were shaken until OD600 = 0.6–0.8. The cultures were centrifuged at 6000 rpm for 10 min to collect the colonies, which were then resuspended in freshly prepared buffer solution and mixed in a 1:1 ratio. After thorough mixing and activation for 3 h, the bacterial suspension was injected into N. tabacum leaves using a 1 mL syringe. Following two days of dark treatment, the injected leaves were collected and examined under an LSM710 laser scanning confocal microscope (Beituo Science, Guangzhou, China) to observe the fluorescent signal, with pSuper1300-GFP serving as the control.
Using CE Design V1.04 software, specific primers were designed (Supplementary Materials Table S1). The pGBKT7-OfERF17 fusion vector was constructed using osmanthus cDNA as the template. The pGBKT7 vector was double-digested at EcoRI and SmaI restriction sites. Subsequently, the digested vector was linked to the target gene and introduced into E. coli DH5α cells. Positive colonies were screened using PCR, and correctly identified colonies were sent to Qingke Biotechnology Company for sequencing. The sequencing results are presented in Supplementary Materials Figure S2. The AH109 yeast strain was transformed with both the recombinant plasmids and the pGBKT7 vector alone (devoid of target genes), where the latter acted as a negative control. Single colonies were selected for positive PCR screening, and those with correct bands were cultured in SD/-Trp broth. A single colony was cultured until the OD600 reached 0.5, and 5 µL of the bacterial suspension was spotted onto SD/-Trp, SD/-Trp-Ade, and SD/-Trp-Ade + X-α-gal media. The plates were incubated at 30 °C in an inverted position, and growth was observed and photographed after five days.

2.6. Agrobacterium-Mediated Transient Overexpression of O. fragrans

To investigate the role of the OfERF17 gene in the aging process of O. fragrans, we employed the Agrobacterium-mediated vacuum infiltration technique to transform petals at the flowering stage. Petals transformed with pSuper1300-OfERF17 served as the experimental group, while those transformed with the empty pSuper1300 vector acted as the control. After light culture, phenotypic changes were observed and recorded. Both groups were subjected to dark culture treatment, and samples were captured at 0 h, 12 h, 24 h, 36 h, and 48 h time points. After 48 h of dark culture, the samples were preserved in liquid nitrogen at a temperature of −80 °C for preservation.

2.7. Agrobacterium-Mediated Overexpression of Nicotiana tabacum

Bacterial suspensions were prepared from Agrobacterium cultures containing pSuper1300-OfERF17 and pSuper1300, then plated on solid medium supplemented with kanamycin (Kana) and incubated at 28 °C for two days with the medium inverted. Individual colonies were picked and cultivated in liquid medium supplemented with kanamycin, and then incubated at 28 °C with agitation at 200 rpm until the optical density (OD) attained 0.8. The bacterial cells were collected, resuspended in double-distilled water (ddH2O), and activated by shaking for three hours. Leaves of wild-type N. tabacum ‘K326’ were washed for over 30 min, surface-sterilized in a laminar flow hood, and cut into 0.5 cm × 0.5 cm squares. The leaf squares were immersed in the target gene solution and the empty bacterial solution, respectively, for 8 min, then dried and placed on symbiotic medium. The plates were incubated in a 25 °C growth chamber. After 3 days of dark treatment, the leaves were transferred to screening medium for 1–2 weeks, followed by secondary screening medium, with subculturing every 2 weeks until regenerated shoots appeared. The regenerated shoots were excised and placed on seedling screening medium for approximately one week. When the shoots grew to over 1 cm, callus from the base was transferred to rooting medium containing antibiotics. Six rooted transgenic seedlings were transplanted into individual pots and grown in a growth chamber. Three seedlings were sampled and stored at −80 °C (Figure 2), while the other three were used to observe the flowering period. For each plant, three replicate experiments were conducted, and images were captured accordingly.

2.8. Physiological Indicators and Quantitative Real-Time Analysis

In this study, we analyzed physiological indicators in 48 h transient O. fragrans petals and S4 N. tabacum petals using a malondialdehyde (MDA) and hydrogen peroxide (H2O2) content assay kit (Beijing Box Raw Technology Co., Ltd., Beijing, China). For each assay, 0.1 g of the petal sample was blended with extraction buffer at a 1:10 ratio in an ice bath. Subsequently, the mixture underwent centrifugation at 4 °C and 8000× g for a duration of 10 min. The resultant supernatant was collected on ice for further analysis, and absorbance measurements were conducted as per the manufacturer’s instructions. To ensure precision, three technical replicates were executed for each sample.
Following the transient infection of O. fragrans petals and heterologous transformation of N. tabacum, we designed fluorescence quantitative primers for senescence marker genes using Primer Premier 5 software (Supplementary Materials Table S1). The 2−ΔΔCt method was employed to compute the relative expression levels of these genes [28], and we subsequently analyzed the expression patterns of OfSAG21, OfACO3, NtSAG12, and NtACO1.

2.9. Data Analysis

The data analysis was conducted using SPSS version 26. To compare differences between two groups, an independent-samples t-test was employed. For comparisons involving more than two groups, a one-way ANOVA was utilized, followed by Duncan’s multiple range test to determine significance. The results are reported as the mean ± standard error (SE) of three independent replicates. Data visualization was carried out using Microsoft Excel 2016.

3. Results

3.1. Expression Analysis of OfERF17 Genes in Different Flowering Times

Utilizing prior transcriptomic data pertaining to O. fragrans senescence, we examined the expression patterns of the ERF transcription factor family throughout five distinct flowering phases: the bud–pedicel phase (Stage 1), bud–eye phase (Stage 2), early flowering phase (Stage 3), peak flowering phase (Stage 4), and terminal flowering phase (Stage 5). The expression of OfERF17 increased significantly during the S4–S5 period, reaching three times the level observed in the S1 period. This expression pattern was validated by qRT-PCR, suggesting that OfERF17 primarily functions during the S5 period and may play a role in regulating O. fragrans petal senescence (Figure 3).

3.2. Bioinformatic Analysis of the OfERF17

Bioinformatic examination of the OfERF17 gene sequence identified a complete open reading frame (ORF) spanning 624 bp, which codes for 207 amino acids. The estimated molecular weight of OfERF17 is 23.07 kDa, with a calculated isoelectric point (pI) of 4.72 and an instability index of 60.8. The NCBI conserved domain map indicates that OfERF17 contains a typical AP2 conserved domain, confirming its classification as a member of the OfERF family (Figure 4A). Secondary structure prediction revealed that OfERF17 consists of 19.81% α-helices, 15.94% extended chains, and 64.25% random coils (Figure 4B). Hydrophilicity analysis classified OfERF17 as a hydrophilic protein (Figure 4C). Transmembrane domain analysis confirmed the absence of a transmembrane structure (Figure 4D). Phosphorylation site prediction identified 18 serine (Ser), 5 threonine (Thr), and 4 tyrosine (Tyr) phosphorylation sites (Figure 4E).
Amino acid sequences of OfERF17 orthologs from various species were obtained from the NCBI database. Phylogenetic tree analyses of amino acid sequences from 10 plant species, including O. fragrans OfERF17 and Olea europaea subsp. europaea, revealed that OfERF17 clusters in the same branch as Olea europaea var. Sylvestris, O. europaea subsp. europaea, Fraxinus pennsylvanica, Forsythia ovata, and Abeliophyllum distichum, all of which belong to the Oleaceae family. OfERF17 is most closely related to O. europaea var. sylvestris and distantly related to Phtheirospermum japonicum and Rehmannia glutinosa (Figure 5A). Multiple sequence alignment of the homologous ERF17 amino acid sequences demonstrated conservation across species, with the highest similarity to O. europaea var. sylvestris (Figure 5B).

3.3. OfERF17 Construction of the Overexpression Vector

Using cDNA from O. fragrans petals as a template, PCR-amplified bands were recovered (Figure 6A). These bands were ligated into the pSuper1300 vector and transformed into E. coli DH5α. Positive clones were sequenced, and sequence alignment using BioXM 2.6 software confirmed consistency with the original sequence, indicating successful construction of the pSuper1300-OfERF17 overexpression vector. The vector was then introduced into Agrobacterium GV3101, and PCR with specific primers identified seven positive clones with consistent band lengths (Figure 6B), confirming successful transformation. This enables the subsequent functional verification of the OfERF17 gene.

3.4. Subcellular Localization and Transcriptional Activation Analysis

An initial assessment of OfERF17’s subcellular localization suggested nuclear localization for the protein. To validate this finding, a fusion construct pSuper1300-OfERF17-GFP was transiently introduced into N. benthamiana leaf cells via Agrobacterium-facilitated transformation. Laser confocal scanning microscopy revealed that both the pSuper1300-OfERF17-GFP fusion vector and the pSuper1300-GFP control exhibited green fluorescent signals in the nucleus. The fluorescence of pSuper1300-OfERF17-GFP completely overlapped with the H2B-mCherry nuclear protein marker (Figure 7A), confirming the nuclear localization of OfERF17. Additionally, yeast transcription autoactivation assays demonstrated that while the pGBKT7 negative control grew only on SD/-Trp medium, the pGBKT7-OfERF17 construct grew on both SD/-Trp and SD/-Trp-Ade media and exhibited a blue coloration on SD/-Trp-Ade medium containing X-α-gal (Figure 7B), indicating that OfERF17 possesses transcriptional autoactivation activity.

3.5. Overexpression of OfERF17 Delays Senescence of O. fragrans Petals

To explore the role of the OfERF17 gene in O. fragrans, we transiently infected flowering petals using the Agrobacterium-mediated method and recorded phenotypic observations (Figure 8A). The results show that, within 48 h after the infection, the degree of petal senescence was not very different between pSuper1300-OfERF17 and control pSuper1300. Compared to the control, OfERF17 expression was upregulated approximately 140-fold. Additionally, the relative expressions of senescence marker gene OfSAG21 and ethylene biosynthesis pathway gene OfACO3 were significantly reduced in the pSuper1300-OfERF17 group compared to the control (Figure 8B,D). Furthermore, MDA and H2O2 content in O. fragrans petals were measured, showing significantly lower levels in the OfERF17 overexpression group versus the control (Figure 8E,F). These findings imply that OfERF17 contributes to the postponement of petal senescence in O. fragrans.

3.6. Overexpression of OfERF17 Delays the Senescence of Nicotiana tabacum Flower Petals

To further investigate the function of OfERF17, we conducted heterologous transformation experiments in N. tabacum. The PCR gel diagram of positive seedlings can be seen in Supplementary Materials Figure S3. During flowering, we recorded the aging process of individual petals in transgenic and wild-type plants (Figure 9A). The results show that transgenic N. tabacum flowers exhibited a one-day delay in senescence compared to wild-type flowers. We observed three wild-type and three transgenic plants, with three petals per plant, and statistically analyzed the flowering duration. Transgenic OfERF17 N. tabacum exhibited significantly longer flowering periods than wild-type plants, with a statistically significant difference (Figure 9E). Additionally, we collected five-period samples for qRT-PCR analysis, revealing that OfERF17 expression peaked during the S4 stage, likely due to species-specific differences in expression trends (Figure 9B). The levels of senescence-associated genes NtSAG12 and NtACO1 were significantly lower in transgenic plants compared to wild-type, with the most pronounced differences observed during these stages (Figure 9C,D). Furthermore, MDA and H2O2 contents in the S4 stage were significantly lower in transgenic plants compared to wild-type (Figure 9F,G), indicating that OfERF17 has a vital function in postponing petal senescence in N. tabacum.

4. Discussion

The ERF gene family is essential for plant growth, development, and responses to abiotic stresses in diverse species. For instance, the genome-wide characterization and expression profiling of the AP2/ERF gene family has been performed in Allium sativum [29], Pinus massoniana [30], Prunus avium [31], and Morus alba [32], providing a theoretical foundation for subsequent functional studies. In A. thaliana, LkERF-B2 enhances salt tolerance [33], while in Phalaenopsis, PeERF1 regulates flower development [34]. Similarly, in O. fragrans, AP2/ERF gene family analysis suggests that certain ERF genes may regulate petal senescence [24]. In this study, OfERF17 exhibited significantly increased expression during the S4-S5 stages, indicating its potential role in petal senescence. The function of ERF17 homologs has been explored in other plants: PpERF017 regulates fruit development [35], CmoERF017 overexpression enhances cold tolerance in N. tabacum [36], and MdERF017 improves iron deficiency tolerance in M. pumila callus [37]. However, the specific function of OfERF17 in O. fragrans remains unclear, prompting its selection for functional validation. Additionally, phosphorylation site modifications have been shown to influence plant traits; for example, dephosphorylated Thr-825 in SlBRI1 increases yield and size in S. lycopersicum [38], while dephosphorylated Ser-1040 enhances heat tolerance [39]. In this study, multiple serine, threonine, and tyrosine phosphorylation sites were predicted in OfERF17, offering future avenues to explore their biological functions in O. fragrans.
Senescence-associated genes (SAGs) play a critical role in the aging of floral organs in plants. For instance, in Paeonia suffruticosa, the expression of IpSAG12 significantly increases during senescence, and silencing IpSAG12 prolongs P. suffruticosa senescence, indicating its positive regulatory role in flower aging [40]. Similarly, NtSAG12 is associated with leaf and flower senescence in N. tabacum, with higher expression observed during leaf senescence [41]. The SAG12 gene has also been used as a senescence marker in other plants. For example, in Gentiana scabra, the CRISPR/Cas9-mediated knockout of EPH1L resulted in delayed flower senescence and reduced expression of the marker gene SAG12 [42]. In this study, NtSAG12 expression was significantly reduced in N. tabacum lines overexpressing OfERF17, suggesting that OfERF17 delays floral senescence. This finding aligns with studies in A. thaliana, where the overexpression of TgWRKY75 increased AtSAG12 expression and promoted leaf senescence [43]. During the five flowering stages of O. fragrans, OfSAG12 was not expressed, while OfSAG21 expression gradually increased with aging. Since AtSAG21 is highly expressed during leaf senescence in A. thaliana [44], we selected OfSAG21 as a senescence marker for OfERF17 functional validation. The overexpression of OfERF17 resulted in lower OfSAG21 expression compared to the control, indicating delayed petal senescence. Additionally, ACC-oxidase (ACO) is a key enzyme in ethylene biosynthesis, playing a significant role in fruit ripening and flower senescence [45,46]. In Petunia hybrida, ethylene treatment induced the specific expression of PhACO1 and PhACO3 in different floral organs [47], while in swallow grass, ethylene treatment accelerated aging with DgACO3 expression exceeding that of natural aging [48]. In this study, OfERF17 overexpression inhibited OfACO3 expression in O. fragrans petals. Similarly, silencing the PDS gene in S. lycopersicum delayed ripening and reduced the expression of ethylene synthesis genes such as ACO3 and ACO1 [49]. These findings suggest that OfERF17 overexpression delays petal senescence in O. fragrans. Furthermore, OfERF17 overexpression in N. tabacum resulted in lower NtACO1 expression compared to wild-type plants, consistent with studies showing that Sl-PI3K overexpression in N. tabacum increased NtACO1 expression and accelerated senescence [50].
Reactive oxygen species (ROS) are critical signaling molecules in plant senescence. Excessive ROS accumulation damages macromolecules and cell membranes, increasing plant vulnerability to oxidative stress [51]. ROS primarily include superoxide radicals (O2−) and H2O2, with H2O2 being more stable and commonly used as a physiological indicator of plant aging [52]. In this study, the overexpression of OfERF17 in O. fragrans and N. tabacum reduced H2O2 content, consistent with findings for Dendrobium nobile, where increased H2O2 levels accelerated flower senescence [53]. MDA, a marker of cell damage, has been used to study hydrogen sulfide’s effect on senescence in lily and hibiscus cut flowers [54]. Lower MDA accumulation in R. rugosa delays cut flower longevity [55], while reduced H2O2 and MDA levels enhance antioxidant capacity in Dianthus cut flowers [56]. In this study, the MDA contents in O. fragrans and N. tabacum overexpressing OfERF17 were significantly lower than in the control, consistent with findings in Brassica rapa, where reduced MDA levels delayed leaf senescence [57]. These results confirm that H2O2 and MDA are key physiological indicators of aging.
Currently, successful genetic transformation in woody plants remains limited. Most studies rely on model plants such as N. tabacum, A. thaliana, and S. lycopersicum for heterologous stable transformation to validate gene functions [58,59,60]. In this study, we established a heterologous transformation system in N. tabacum to verify the function of OfERF17 in overexpression lines. Compared to heterologous transformation, transient transformation systems offer a simpler and faster method for gene function validation [61]. In recent years, transient transformation systems have been developed in Ginkgo biloba [62], Pinus tabuliformis [63], Cinnamomum camphora [64], and other species. For example, in Paeonia suffruticosa, the transient infection of petals via Agrobacterium-mediated methods was used to compare senescence levels between overexpression and control groups, providing preliminary functional validation [65]. The transient transformation system in O. fragrans is relatively well-established [66,67,68,69]. In this study, we constructed the pSuper1300-OfERF17 fusion vector using Agrobacterium-mediated methods and demonstrated that OfERF17 overexpression delays flower senescence. Additionally, callus genetic transformation systems have been widely applied in Prunus persica [70], Vernicia fordii [71], and Pyrus armeniacaefolia [72]. Although a callus genetic transformation system has not yet been reported for O. fragrans, transcriptomic and endogenous hormone analyses of callus tissues [73] provide a foundation for its development. Future efforts will focus on exploring genetic transformation systems to facilitate the cultivation of improved varieties.

5. Conclusions

In this study, based on previous transcriptomic data analyses, we hypothesized that the OfERF17 gene might regulate flower senescence in O. fragrans. To investigate this, we conducted bioinformatics analyses and successfully cloned OfERF17. We then performed transient infection experiments in O. fragrans and heterologous transformation experiments in N. tabacum to validate its function. The results demonstrate that OfERF17 delays flower senescence and extends the full-bloom period. These findings confirm the functional role of OfERF17, providing a theoretical foundation for breeding new varieties and advancing the study of flower senescence mechanisms in O. fragrans.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16040615/s1, Table S1: The primers used in this article; Figure S1: Plot of the E. coli DH5α sequencing alignment of pSuper1300-OfERF17; Figure S2: Plot of the E. coli DH5α sequencing alignment of pGBKT7-OfERF17; Figure S3: PCR gel diagram of transgenic positive seedlings.

Author Contributions

Conceptualization, G.C. and L.W.; methodology, G.C.; software, D.Z.; validation, D.Z., F.C. and Y.Z.; formal analysis, D.Z.; investigation, H.G.; resources, L.W. and X.Y.; data curation, H.G.; writing—original draft preparation, D.Z., F.C. and Y.Z.; writing—review and editing, X.Y., L.W. and Y.Y.; visualization, X.Q.; supervision, X.Y.; project administration, G.C.; funding acquisition, G.C. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Youth Science Fund Project of the National Natural Science Foundation of China (grant number 32201625), under the project “Investigating the Molecular Mechanism of Ethylene-Responsive Transcription Factor OfERF106 in Regulating Petal Senescence in O. fragrans”; by the Jiangsu Province Higher Education Basic Science (Natural Science) General Project (grant number 21KJB220006), under the project “Screening, Identification, and Functional Characterization of Proteins Interacting with Aging-Related Transcription Factor OfERF3 in O. fragrans”; and by the Jiangsu Forestry Bureau (grant number LYKJ [2020]26), through the Provincial Long-Term Scientific Research Base for Color Leaf Tree Breeding and Cultivation.

Data Availability Statement

All data are available upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Flower morphology of five flowering periods. S1: bud—pedicel phase. S2: bud—eye phase. S3: early flowering phase. S4: peak flowering phase. S5: terminal flowering phase.
Figure 1. Flower morphology of five flowering periods. S1: bud—pedicel phase. S2: bud—eye phase. S3: early flowering phase. S4: peak flowering phase. S5: terminal flowering phase.
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Figure 2. Five flower morphological patterns of large flower N. tabacum. S1: tight bud phase. S2: mature bud phase. S3: early flowering phase. S4: peak flowering phase. S5: terminal flowering phase.
Figure 2. Five flower morphological patterns of large flower N. tabacum. S1: tight bud phase. S2: mature bud phase. S3: early flowering phase. S4: peak flowering phase. S5: terminal flowering phase.
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Figure 3. Expression levels of OfERF17 at five flowering times. Different letters indicate a significant difference p < 0.05, and having the same letters indicates no significant difference.
Figure 3. Expression levels of OfERF17 at five flowering times. Different letters indicate a significant difference p < 0.05, and having the same letters indicates no significant difference.
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Figure 4. Bioinformatics analysis of OfERF17. (A) Conserved domain prediction of OfERF17. (B) Secondary structure prediction of the OfERF17-encoded protein, blue lines for α-helices, purple lines for extended strands, and yellow lines for random coil. (C) OfERF17 Hydrophilicity and hydrophobicity prediction of the encoding proteins. (D) Transmembrane domain analysis of OfERF17. The blue line indicates the probability within the membrane in the ordinate, the thin yellow line shows the probability outside the membrane in the ordinate, and the yellow thick line shows the area outside the membrane in the abscissa. (E) Analysis of the phosphorylation sites of the protein encoded by OfERF17.
Figure 4. Bioinformatics analysis of OfERF17. (A) Conserved domain prediction of OfERF17. (B) Secondary structure prediction of the OfERF17-encoded protein, blue lines for α-helices, purple lines for extended strands, and yellow lines for random coil. (C) OfERF17 Hydrophilicity and hydrophobicity prediction of the encoding proteins. (D) Transmembrane domain analysis of OfERF17. The blue line indicates the probability within the membrane in the ordinate, the thin yellow line shows the probability outside the membrane in the ordinate, and the yellow thick line shows the area outside the membrane in the abscissa. (E) Analysis of the phosphorylation sites of the protein encoded by OfERF17.
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Figure 5. OfERF17 phylogenetic tree and multiple sequence alignments. (A) The phylogenetic tree of OfERF17 and other species. Triangle symbols highlight the OfERF17. (B) The amino acid sequence of OfERF17 was aligned with ERF17 sequences from other species. Dark blue shading denotes 100% sequence identity, pink shading represents sequence similarity ranging from 100% to 75%, and light blue shading indicates sequence similarity between 75% and 50%.
Figure 5. OfERF17 phylogenetic tree and multiple sequence alignments. (A) The phylogenetic tree of OfERF17 and other species. Triangle symbols highlight the OfERF17. (B) The amino acid sequence of OfERF17 was aligned with ERF17 sequences from other species. Dark blue shading denotes 100% sequence identity, pink shading represents sequence similarity ranging from 100% to 75%, and light blue shading indicates sequence similarity between 75% and 50%.
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Figure 6. OfERF17 PCR glue diagram. (A) OfERF17 clone glue, M is DL2000 marker (TaKaRa, Dalian, China). a is the clonogenic band. (B) PCR gel diagram of pSuper1300-OfERF17 Agrobacterium, 1–7 for positive bacteria and M for DL2000 marker.
Figure 6. OfERF17 PCR glue diagram. (A) OfERF17 clone glue, M is DL2000 marker (TaKaRa, Dalian, China). a is the clonogenic band. (B) PCR gel diagram of pSuper1300-OfERF17 Agrobacterium, 1–7 for positive bacteria and M for DL2000 marker.
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Figure 7. OfERF17 Subcellular localization map and yeast transcription autoactivation verification map. (A) Subcellular localization map, where pSuper1300-GFP is the yeast transcription autoactivation verification map of empty load control; (B) where pGBKT7 is the negative control.
Figure 7. OfERF17 Subcellular localization map and yeast transcription autoactivation verification map. (A) Subcellular localization map, where pSuper1300-GFP is the yeast transcription autoactivation verification map of empty load control; (B) where pGBKT7 is the negative control.
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Figure 8. The overexpression of OfERF17 delays the senescence of O. fragrans petals. (A) The senescence phenotypes of pSuper1300 control and OfERF17 overexpressed O. fragrans petals at 0 h, 12 h, 24 h, 36 h and 48 h. (B) Expression of OfERF17 in the petals. (C,D) Expression levels of senescence marker genes in the petals of O. fragrans with pSuper1300 serving as the control and OfERF17 overexpression. (E,F) pSuper1300 MDA and H2O2 in control and OfERF17. * Denotes a statistically significant difference (p < 0.05). ** Signifies a very significant difference (p < 0.01). *** Indicates an extremely significant difference (p < 0.001).
Figure 8. The overexpression of OfERF17 delays the senescence of O. fragrans petals. (A) The senescence phenotypes of pSuper1300 control and OfERF17 overexpressed O. fragrans petals at 0 h, 12 h, 24 h, 36 h and 48 h. (B) Expression of OfERF17 in the petals. (C,D) Expression levels of senescence marker genes in the petals of O. fragrans with pSuper1300 serving as the control and OfERF17 overexpression. (E,F) pSuper1300 MDA and H2O2 in control and OfERF17. * Denotes a statistically significant difference (p < 0.05). ** Signifies a very significant difference (p < 0.01). *** Indicates an extremely significant difference (p < 0.001).
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Figure 9. The overexpression of OfERF17 delays the senescence of large N. tabacum petals. (A) Wild-type and OfERF17 overexpress the senescence phenotype of large N. tabacum petals. (B) Expression of OfERF17 in the petals of large N. tabacum. (C,D) Expression of senescence genes in overexpressed wild type and OfERF17. (E) Flowering time of wild-type and OfERF17-overexpressing flowering N. tabacum in the S4 period. (F,G) H2O2 and MDA in the petals of wild-type–overexpressing and OfERF17-growing N. tabacum. Different letters indicate a significant difference p < 0.05, and having the same letters indicates no significant difference. * Denotes a statistically significant difference (p < 0.05). ** Signifies a very significant difference (p < 0.01).
Figure 9. The overexpression of OfERF17 delays the senescence of large N. tabacum petals. (A) Wild-type and OfERF17 overexpress the senescence phenotype of large N. tabacum petals. (B) Expression of OfERF17 in the petals of large N. tabacum. (C,D) Expression of senescence genes in overexpressed wild type and OfERF17. (E) Flowering time of wild-type and OfERF17-overexpressing flowering N. tabacum in the S4 period. (F,G) H2O2 and MDA in the petals of wild-type–overexpressing and OfERF17-growing N. tabacum. Different letters indicate a significant difference p < 0.05, and having the same letters indicates no significant difference. * Denotes a statistically significant difference (p < 0.05). ** Signifies a very significant difference (p < 0.01).
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Chen, G.; Zhang, D.; Chen, F.; Zhou, Y.; Gu, H.; Qin, X.; Yue, Y.; Wang, L.; Yang, X. Characterization of OfERF17 as a Key Regulator of Petal Senescence in Osmanthus fragrans. Forests 2025, 16, 615. https://doi.org/10.3390/f16040615

AMA Style

Chen G, Zhang D, Chen F, Zhou Y, Gu H, Qin X, Yue Y, Wang L, Yang X. Characterization of OfERF17 as a Key Regulator of Petal Senescence in Osmanthus fragrans. Forests. 2025; 16(4):615. https://doi.org/10.3390/f16040615

Chicago/Turabian Style

Chen, Gongwei, Dandan Zhang, Fengyuan Chen, Yixiao Zhou, Heng Gu, Xuyang Qin, Yuanzheng Yue, Lianggui Wang, and Xiulian Yang. 2025. "Characterization of OfERF17 as a Key Regulator of Petal Senescence in Osmanthus fragrans" Forests 16, no. 4: 615. https://doi.org/10.3390/f16040615

APA Style

Chen, G., Zhang, D., Chen, F., Zhou, Y., Gu, H., Qin, X., Yue, Y., Wang, L., & Yang, X. (2025). Characterization of OfERF17 as a Key Regulator of Petal Senescence in Osmanthus fragrans. Forests, 16(4), 615. https://doi.org/10.3390/f16040615

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