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. 2026 Feb 14;15(4):610. doi: 10.3390/plants15040610

Cloning, Expression and Functional Study of OfCOR27 Gene in Osmanthus fragrans

Ruiqi Chen 1,†, Jinfeng Li 1,†, Shenglian Li 1,†, Daowu Zhang 1, Min Zhang 1, Yifan Duan 1,*
Editor: Jiangqi Wen1
PMCID: PMC12943921  PMID: 41754316

Abstract

Blooming time is an important basis for constructing plant landscapes. The short flowering period of Osmanthus fragrans, recognized as one of the ten traditional flowers in China, considerably constrains the further utilization of its resources. To clarify O. fragrans flowering regulation, this study focused on OfCOR27, conducting cloning, expression analysis, and functional verification to explore its effects on O. fragrans flowering time. A COR27 phylogenetic tree was built across six species; OfCOR27 physicochemical properties, conserved structures, and promoter cis-elements were analyzed. OfCOR27 CDS was cloned, fusion vectors were transformed into Nicotiana benthamiana, and organ-specific expression was tested in two O. fragrans cultivars. Overexpression vectors were transformed into Arabidopsis thaliana, with qRT-PCR verifying gene function. Five OfCOR27s were identified, showing evolutionary conservation. OfCOR27, which localizes to the nucleus and is associated with flowering regulation, shows higher expression in ‘Sijigui’ than in ‘XiaoyeSugui’. Overexpression of OfCOR27 promoted flowering in A. thaliana, whereas the AtCOR27 mutant flowered later. This confirms OfCOR27 is a positive regulator of plant flowering, which may promote flowering by enhancing the expression of flowering-promoting genes and altering hormone levels, providing a theoretical basis and candidate gene for the genetic improvement of flowering traits in woody ornamental plants.

Keywords: COR27 gene, Osmanthus fragrans, flowering regulation, Arabidopsis thaliana

1. Introduction

Blooming time is an important basis for constructing plant landscapes, as it directly determines the ornamental value of plants and their seasonal sequence in scenic composition [1]. It is well exemplified by ornamental trees such as those in the Osmanthus genus, whose distinct flowering period is a central feature of autumn landscapes. The development of such ornamental traits is governed by complex molecular mechanisms that regulate flowering in plants [2]. Many genes that regulate blooming have been found in the model plant Arabidopsis thaliana [3,4]. These genes function by integrating external signals like photoperiod and temperature, which then guide an internal gene regulatory network to determine the timing of flowering [5]. However, the precise improvement of these valuable ornamental traits is constrained by the sheer complexity of the underlying molecular networks.

COR27 (COLD-REGULATED GENE 27) is a member of the CCR (Cold and Clock Regulated) family. Initially, it was identified in transcriptome analyses of cells under low-temperature conditions [6]. Studies have shown that the expression of both COR27 and COR28 can be rapidly stimulated by low temperatures, and they were named based on this characteristic together with the molecular weight of their encoded proteins. In A. thaliana, COR27 was further characterized as a cold stress-responsive gene, encoding a small molecular weight protein induced by cold stress and acting as a critical component in the plant’s adaptation to low temperatures [7]. Evidence for the conserved function of COR27 comes from research in diverse plant species. In grapevine, cold resistance evaluation of three VvCOR27-overexpressing transgenic Arabidopsis lines demonstrated that VvCOR27 participates in the plant cold stress response and acts as a positive regulator to enhance cold tolerance [8].

Research has further revealed that COR27 serves as a regulatory hub, integrating environmental signals to control plant development. This function is demonstrated in its regulation of photoperiod and the circadian clock. Besides their response to low temperature, COR27 and its homolog COR28 also respond to blue light. The mutants of both genes exhibit delayed flowering under normal conditions yet enhanced cold tolerance under stress [9], identifying COR27 as a key integrator of photoperiod and low-temperature signals for flowering time regulation.

Later studies found that mutations in COR27 and COR28 lead to a longer period of core circadian oscillator oscillations. The underlying mechanism was elucidated by EMSA, ChIP, and transcriptome analyses, which showed that the core clock component CCA1 binds to the EE elements in COR27/28 promoters and regulates their rhythmic expression. This research confirmed the role of COR27 in promoting flowering [10]. It was also discovered that COR27/28 transcripts are suppressed by blue light, yet the proteins are degraded in the dark via the COP1-SPA1 complex. This degradation, coupled with its dual role in inhibiting HY5 and promoting PIF4 expression, allows COR27 to connect the circadian clock to light signaling, ultimately establishing its function as a negative regulator of photomorphogenesis [11]. Notably, while these studies have clearly demonstrated the flowering-promoting function of COR27 in the herbaceous model plant A. thaliana, the functional characterization of COR27 homologs in woody plants—especially in ornamental woody species with unique flowering traits such as O. fragrans—remains largely unexplored. This represents a significant knowledge gap, as woody plants often exhibit distinct flowering characteristics (e.g., perennial growth cycle, cultivar-specific flowering frequency) that differ fundamentally from those of annual herbaceous plants like A. thaliana. Whether COR27 homologs in O. fragrans retain the conserved flowering-promoting function, and more importantly, whether they are involved in regulating the specific flowering traits of O. fragrans remains unknown.

It has been further established that COR27 and COR28 act by embedding themselves in the COP1-HY5 core network, integrating light and cold temperature signals to regulate photomorphogenesis [12]. In addition to their interaction with COP1, COR27/28 also interact with the upstream light signaling elements phyA and phyB [13]. This indicates an additional layer of interaction, implicating COR27/28 in having broader regulatory functions in the light signaling network. COR27 thus serves as a key signaling node, responding not only to cold but also functioning in light signaling, thereby regulating seedling photomorphogenesis through the integration of environmental cues. The exposure of plants to appropriate daylengths typically triggers flowering, a process which uses the circadian clock to measure time [14]. Research has also shown that the circadian clock itself is regulated by temperature [15]. By binding to the chromatin of dark-associated genes, COR27 and COR28 function as negative regulators of cold tolerance. They modulate the central circadian clock, thereby promoting flowering [2]. This regulatory activity leads to COR27 upregulation, which in turn promotes flowering initiation, accelerates leaf formation, and advances physiological age during vernalization [16]. To conclude, COR27 acts as a key signaling integrator whose dynamic regulation reflects multiple environmental inputs. The protein’s abundance and activity are modulated by cold, light, and circadian signals, while its function is regulated by the COP1-HY5 pathway. This positions COR27 to precisely coordinate critical plant adaptations—including cold tolerance, photomorphogenic development, and flowering timing—across multiple regulatory layers.

Osmanthus fragrans Lour. is an evergreen tree or shrub that belongs to the genus Osmanthus and the Oleaceae family [17], and boasts a cultivation history in China exceeding 2500 years. Renowned for its delightful fragrance and superior ornamental qualities, it is widely cultivated in landscapes across northern and southern China [18] and is a culturally significant species, counted among China’s top ten traditional flowers, holding significant ornamental and economic value. Research on O. fragrans covers various aspects, focusing notably on ornamental traits such as flower color, which varies richly among cultivars [19]. These colors—yellow, orange, and red—are mainly determined by carotenoid content, especially β-carotene [20,21]. Its intense fragrance is primarily shaped by constituents including linalool, β-ionone, and α-ionone. Furthermore, the diversity in color and aroma is determined by the quantities of carotenoids and terpenoids, with the accumulation of these specific compounds being the key factor [22,23]. Advances have also been made in understanding petal senescence in O. fragrans. This process is associated with a rise in ethylene levels [24]. Additionally, the expression of genes like OfERFs, which peaks during flowering and then decreases, suggests a regulatory role in senescence [25].

Despite advances in color, scent, and senescence studies, the short flowering period of O. fragrans considerably constrains the further utilization of its resources, while molecular-level insights into its regulation are still in their infancy. Consequently, the elucidation of the related molecular mechanisms governing its flowering is extremely significant for its production and application. Previous studies have confirmed that the COR27 positively regulates flowering time in A. thaliana; but its function in O. fragrans is yet to be explored. Therefore, this study aims to clone OfCOR27 and conduct a functional characterization to investigate its potential role in regulating the floral phenology of O. fragrans. To clarify the experimental design rationale, we specifically selected target genes for expression analysis based on the following considerations. We chose key marker genes from the A. thaliana flowering regulation pathway, including AtFT (a flowering signal integrator) [26], AtLFY (a determinant of floral meristem identity) [27], and AtSOC1 (a critical downstream regulator in the flowering pathway) [28]. The functions of these genes are well-established in model plants, and examining their expression changes can directly reflect the impact of OfCOR27 on the conserved core flowering pathway. Based on the existing literature indicating close associations between O. fragrans flowering and gibberellin (GA) as well as abscisic acid (ABA) signaling, we selected AtGA3ox1 (a key enzyme gene for GA synthesis) [29] and AtABA2 (a key enzyme gene for ABA synthesis) in A. thaliana. Both genes have been confirmed to regulate flowering time in A. thaliana, and analyzing their expression patterns helps clarify the potential relationship between OfCOR27 and hormone pathways.

The innovation of this research is primarily reflected in the following aspects: it extends the functional study of the COR27 gene from herbaceous model plants to the woody ornamental species O. fragrans, providing direct evidence for the functional analysis of this homologous gene within the Osmanthus genus; it further investigates whether OfCOR27 is involved in regulating cultivar-specific flowering traits (such as recurrent flowering), thereby offering clues to understanding the molecular mechanisms underlying unique flowering characteristics in woody plants; meanwhile, the findings may provide theoretical references and genetic resources for molecular breeding of flowering traits in O. fragrans. This work is expected to offer scientific support for the genetic improvement and breeding of new cultivars in O. fragrans.

2. Results

2.1. Identification and Phylogenetic Analysis of COR27 Gene Family in O. fragrans

A combined methodology employing BLASTP, utilizing the A. thaliana AtCOR27 protein sequence against the O. fragrans genome, along with HMMER for conserved domain analysis, successfully identified five OfCOR27 genes. To explore the evolutionary associations, the protein sequences of these OfCOR27s were employed as queries to retrieve highly similar homologous sequences from A. thaliana, Vitis vinifera, Nicotiana tabacum, Olea europaea, and Oryza sativa through NCBI BLAST. Using 1000 bootstrap replications and the neighbor-joining algorithm, phylogenetic analysis of the OfCOR27 gene family protein sequences and homologous sequences was carried out in MEGA11. The findings revealed that the 24 amino acid sequences from the six species were clustered into three distinct clades, designated as COR15A, COR28 and COR27. Significantly, OfCOR27 and evm.model.contig163.14 were grouped within the COR27 clade, which also encompassed AtCOR27 (a known flowering-promoting gene in herbaceous plants) and OsCOR27 (Figure 1a). This phylogenetic positioning confirms that OfCOR27 is orthologous to the COR27 genes of these species, indicating a close evolutionary kinship. Notably, as the first functionally characterized COR27 homolog in the O. fragrans genus, OfCOR27 shares evolutionary conservation with AtCOR27, laying a foundation for verifying its conserved flowering-regulating function in woody plants.

Figure 1.

Figure 1

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Bioinformatic profiling of the COR27 gene family in O. fragrans. (a) Evolutionary tree analysis of COR27 gene family; (b) Analysis of gene structure and conserved motifs of OfCOR27 family members; (c) Sequence alignment of OfCOR27 gene family. Numbers above the alignment indicate residue positions (tens and units). Dots (.) are decade markers; dots within sequences represent alignment gaps; (d) Cis-acting element analysis of the OfCOR27 gene family. ACE: light-responsive element; GARE: gibberellin-responsive element; ABRE: cis-acting element mediating abscisic acid responsiveness; TCA cis-acting element mediating salicylic acid responsiveness; TGA: cis-acting regulatory element governing auxin responsiveness; LTR: cis-acting element mediating low-temperature responsiveness.

2.2. Motif Identification, Gene Structure and Multiple Sequence Alignment Analysis

Analysis of conserved motifs using the MEME suite indicated that the OfCOR27 family exhibits strongly conserved motifs—specifically motif 1, motif 2, and motif 3, which were found in every member (Figure 1b). This ubiquitous conservation of these motifs suggests they play a fundamental and essential role in OfCOR27 gene family function. Moreover, the gene structure of all members consisted of one or more exons without introns, demonstrating structural conservation and suggesting shared features in gene expression regulation. Consistent with this structural conservation, multiple sequence alignment revealed significant sequence identity and similarity within the OfCOR27 gene family (Figure 1c), further supporting the functional conservation of this gene family across evolution.

2.3. Analysis of Cis-Acting Regulatory Elements Within Promoter

One hundred fifteen potential cis-acting regulatory elements were identified in the 2 kb upstream promoter regions of the OfCOR27 gene family; these elements were categorized into six clusters associated with flowering regulation (Figure 1d). They are primarily linked to light responsiveness and phytohormone signaling (encompassing auxin, gibberellin, abscisic acid, and salicylic acid, e.g.). These cis-acting elements provide preliminary clues for the potential regulatory pathways of OfCOR27, but their actual involvement in flowering regulation requires verification through follow-up experiments. Notably, the enrichment of light-responsive and auxin-responsive elements suggests that OfCOR27 may be involved in hormone-mediated flowering regulation in O. fragrans, which aligns with the subsequent functional verification results of its flowering-promoting role.

2.4. Protein Structure Analysis

The OfCOR27 protein’s physicochemical property detection results revealed that it has a molecular weight of 27,787.11 Da, an aliphatic amino acid index of 46.35, an instability index of 50.43 (≥40 indicates instability), a grand average of hydropathicity (GRAVY) of −1.150 (<0 indicates hydrophilicity), and an isoelectric point (pI) of 6.00. Based on these parameters, the OfCOR27 protein can be classified as an unstable and hydrophilic protein (Figure 2a). The secondary structure was predominantly composed of random coils (77.05%), with minor components of α-helix (18.85%) and extended strand (4.10%) (Figure 2b). The prediction of the tertiary structure confirmed a predominantly disordered conformation, which is consistent with the intrinsic disorder characteristic of the COR27 family and supports its potential role as a flexible regulatory protein in signaling pathways (Figure 2c).

Figure 2.

Figure 2

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Protein structure analysis of OfCOR27. (a) OfCOR27 amino acid hydrophilic; (b) Secondary structure of OfCOR27 protein; (c) Tertiary structure of OfCOR27 protein. Prediction confidence: dark blue > light blue > yellow > orange (high to low). Three evolutionarily conserved regions in the OfCOR27 protein sequence are located at amino acid positions 40–74 (CR1), 101–150 (CR2), and 186–228 (CR3). These regions correspond to putative functional modules that are characteristic of the COR27 family.

2.5. Subcellular Localization Analysis of OfCOR27

Subcellular localization was examined via transient expression in N. benthamiana abaxial epidermal cells (three biological replicates, 10 cells observed per replicate). The control empty vector pMDC43-GFP exhibited fluorescent signals within the nucleus and cytoplasm. In contrast, the pMDC43-GFP-OfCOR27 fusion protein was predominantly localized in the nucleus, with no obvious signals in the cytoplasm or cell membrane (Figure 3), consistent with its putative role as a nuclear regulatory protein and aligning with the subcellular distribution of AtCOR27 and other orthologous COR27 proteins reported in previous studies.

Figure 3.

Figure 3

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Subcellular localization of OfCOR27 in N. benthamiana leaf abaxial epidermis. Green fluorescence indicates GFP signals, red fluorescence indicates chloroplast autofluorescence, and the merged panel shows the co-localization of the two signals. Scale Bar = 50 μm.

2.6. Expression Profiling of OfCOR27 Genes

2.6.1. Expression Across Flower Bud Developmental Stages

The flower buds of two O. fragrans cultivars, “Sijigui” (recurrent flowering) and “Xiaoyesugui” (single flowering per year), showed a significant upregulation of OfCOR27 expression, exhibiting distinct organ-specific expression patterns (Figure 4a). Among them, ‘Sijigui’ had a notably higher expression level of OfCOR27 during the bud sprouting stage than ‘Xiaoyesugui’ (p < 0.05), indicating a potential association with its recurrent flowering trait. In ‘Xiaoyesugui’, high expression was detected at the “Yuanzhu” stage, a cultivar-specific phase of bud development. These findings suggest that OfCOR27 participates in the modulation of flowering timing and may impact flowering frequency in “Sijigui”.

Figure 4.

Figure 4

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Comprehensive analysis of COR27 family, flowering, and hormone-related gene expression in O. fragrans flower buds and A. thaliana genetic models. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. (a) qRT-PCR of the OfCOR27. SJG: O. fragrans ‘Sijigui’. XYSG: O. fragrans ‘Xiaoyesugui’; HY1: Sprouting stage; HY2: Germination stage; HY3: Round bead stage; XY: Fragrant eye stage; CH: Initial flowering stage; SH: Full flowering stage; MH: Late flowering stage; NY: New leaves; LY: Old leaves; J: Stem; (b) Expression levels of flowering-related genes in OfCOR27-overexpressing A. thaliana and wild-type A. thaliana; (c) Expression levels of hormone synthesis-related genes in OfCOR27-overexpressing A. thaliana and wild-type A. thaliana; (d) Expression levels of flowering-related genes in AtCOR27 mutant A. thaliana and wild-type A. thaliana; (e) Expression levels of hormone synthesis-related genes in AtCOR27 mutant A. thaliana and wild-type A. thaliana.

2.6.2. Quantification of Flowering-Related Gene Expression and Hormone Synthesis-Related Genes in OfCOR27-Overexpressing A. thaliana

In transgenic A. thaliana overexpressing OfCOR27, key flowering-promoting genes (AtLFY, AtFT, AtAP1, AtFUL) were significantly upregulated, while the flowering repressor AtFLC was downregulated (Figure 4b,c). Genes involved in hormone synthesis were also upregulated, indicating that OfCOR27 may affect flowering and plant growth through hormonal pathways.

2.6.3. Expression in the A. thaliana AtCOR27 Mutant

In the AtCOR27 mutant, expression of flowering-promoting genes (AtSOC1, AtFUL) was reduced, whereas AtFLC was upregulated (Figure 4d,e). Slight downregulation was observed in gibberellin- and auxin-related genes, while ethylene synthesis-related genes were markedly upregulated. These changes are linked to delayed bolting, postponed flowering, and elevated rosette leaf count (Figure 5), validating that AtCOR27 functions in modulating flowering timing and developmental progression.

Figure 5.

Figure 5

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Phenotypic statistics of AtCOR27 mutant A. thaliana. * indicates p < 0.05, ** indicates p < 0.01. (a) Phenotypic comparison between mutant A. thaliana and wild-type A. thaliana; (b) Phenotypic comparison between mutant A. thaliana and wild-type A. thaliana at the late flowering stage; (c) Time taken for mutant A. thaliana and wild-type A. thaliana to bolt to 1 cm in height; (d) Flowering time of mutant A. thaliana and wild-type A. thaliana; (e) Rosette leaf number in wild-type and mutant A. thaliana at flowering.

2.7. Phenotypic Effects of OfCOR27 Overexpression Transgenic Mutants and AtCOR27 Loss-of-Function Mutants

Overexpression of OfCOR27 in A. thaliana resulted in significantly earlier bolting (by 5.79 days) and flowering (by 2.25 days), with a reduction in rosette leaf number (by 0.48), though final leaf count at flowering revealed no discernible variation from the wild type (Figure 6). Conversely, the AtCOR27 mutant exhibited delayed bolting and flowering, and produced more rosette leaves (Figure 5), reinforcing the role of COR27 in promoting flowering and accelerating development.

Figure 6.

Figure 6

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Phenotypic statistics of T2 generation transgenic A. thaliana with OfCOR27 overexpression. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. (a) Phenotypic comparison between OfCOR27-transgenic A. thaliana and wild-type (WT) A. thaliana; (b) Phenotypic comparison between transgenic A. thaliana and wild-type A. thaliana at the early flowering stage; (c) Comparison of the time taken for A. thaliana to bolt to 1 cm in height; (d) Comparison of flowering time in A. thaliana; (e) Comparison of the number of rosette leaves in A. thaliana at flowering.

3. Discussion

3.1. Evolutionary Conservation and Gene Structure of OfCOR27 Gene Family

Five members of the OfCOR27 gene family were found in O. fragrans in the current study. Phylogenetic analysis indicated that OfCOR27 has a close evolutionary relationship with AtCOR27 from A. thaliana and OsCOR27 from O. sativa. It has been previously verified that AtCOR27 in A. thaliana influences flowering time by modulating cold signaling and flowering pathways. Based on this, we speculate that OfCOR27 may have retained a similar functional potential during evolution, which warrants further validation through functional complementation assays. Moreover, in the gene structure analysis, all family members were found to be composed of one or more exons. The conservation of exon number and distribution is generally related to the stability of gene transcriptional regulation [30]. Based on these findings, we speculate that OfCOR27 family genes may maintain relatively stable transcriptional regulatory patterns in O. fragrans. However, their organ-specific and developmental stage-dependent expression profiles require further elucidation through quantitative expression analysis. Thus, this analysis establishes a preliminary link between gene structure and potential functional regulation, providing clues and hypotheses for subsequent functional studies.

3.2. Nuclear Localization and Functional Implications of OfCOR27

In Brassica napus, COR proteins are localized in multiple organelles, encompassing chloroplasts and the plasma membranes of specific organelles. The multi-organelle localization of COR proteins in B. napus might be correlated with their “multifunctionality”, specifically, their concurrent participation in photosynthesis stress responses and flowering time regulation [31]. In A. thaliana, the AtCOR27 protein was predominantly localized in the nucleus. In the current study on O. fragrans, the OfCOR27 protein was also discovered to be mainly distributed in the nucleus. Moreover, the overexpression of OfCOR27 in A. thaliana notably upregulated the expression of the nuclear-localized AtLFY and AtFT. This suggests that the nuclear localization of OfCOR27 offers a spatial foundation for its regulation of flowering-related genes in the nucleus. The “localization-function” relationship between the two is highly congruent, which also implies that OfCOR27 may adhere to a nuclear transcriptional regulation mechanism analogous to that of AtCOR27. In the present research, the localization of OfCOR27 was solely examined in the leaf cells of N. benthamiana, and its localization alterations during different developmental stages of O. fragrans flower buds were not investigated. The dynamic changes in cell structure and function during flower bud development may result in stage-specific disparities in OfCOR27 localization. This dynamic localization characteristic might be associated with the spatiotemporal specificity of flowering time regulation, which necessitates further verification in subsequent investigations.

3.3. Cis-Acting Elements and Hormone Signaling Within OfCOR27 Promoter

The majority of reported COR family members are involved in modulating flowering timing. Cis-elements in gene promoters are recognized to affect growth, development, and stress responses [32,33]. Profiling the cis-acting elements within the OfCOR27 promoter is beneficial for comprehending its potential biological functions. The OfCOR27 promoter is abundant in light-responsive and hormone-responsive elements, implying that OfCOR27 could participate in photoperiod- and hormone-mediated flowering regulation. Gibberellin (GA), a well-established flowering-promoting hormone, has been demonstrated to promote flowering in crops such as Chrysanthemum morifolium [34], V. vinifera [35], and Gossypium hirsutum [36]. Conversely, abscisic acid (ABA) can notably enhance the activity of superoxide dismutase (SOD) and the content of soluble sugars, while alleviating the decrease in chlorophyll content and decelerating plant senescence [37]. Consequently, an increase in ABA content generally postpones flowering.

How does OfCOR27 integrate the “antagonistic effect” between GA and ABA in O. fragrans? The phytohormone regulatory network is highly complex and context-dependent. For instance, not only GA and ABA, but other hormones such as SA and auxin can exhibit diverse promotive or inhibitory effects on flowering across different species, or even within the same species under varying conditions. Salicylic acid (SA) treatment can markedly accelerate flower bud differentiation and promote early flowering in C. morifolium. In Cucumis sativus, low concentrations of auxin can also facilitate the growth of flower buds [38]. Nevertheless, exogenous application of SA in Freesia refracta inhibits the plant’s growth, impacts its corms, and results in delayed flowering [39]. Auxin can either inhibit or promote flower bud differentiation: it inhibits flower bud differentiation in Prunus avium [40], but promotes this process in plants such as Malus pumila [41], Ginkgo biloba [42], and Dianthus chinensis [43]. This discovery indicates that COR genes in O. fragrans and other plant species exhibit functional similarities as they are engaged in plant growth, development, and stress responses. Therefore, based on the presence of hormone-responsive elements such as GARE and ABRE in the OfCOR27 promoter, we hypothesize that OfCOR27 may act as an integrative node involved in balancing GA and ABA signaling transduction, thereby regulating the flowering time in O. fragrans. The specific mechanisms underlying this integration—for instance, whether it involves differential binding to or regulation of distinct cis-elements—will be an important direction for future investigation.

3.4. Organ-Specific Expression of OfCOR27 and Its Association with Cultivar-Specific Flowering Differences in O. fragrans

To more precisely characterize the specific expression pattern of OfCOR27 in O. fragrans, the relative expression levels of six genes across various tissues were analyzed. Through temperature regulation and quantitative real-time polymerase chain reaction (qRT-PCR) analysis, it was discovered that OfTCP5, OfTCP9, and OfTCP12 appear to be the core genes responding to flower bud differentiation in O. fragrans under low-temperature [44] conditions. Transcriptome analysis of O. fragrans flower buds collected over the course of a year identified differentially expressed genes (DEGs). Subsequently, the construction of overexpression vectors confirmed that OfFT is a flowering-promoting gene, while OfBFT is a flowering-repressing gene [45]. The cloning of OfSPL genes and qRT-PCR analysis indicated that low temperature may promote the expression of certain OfSPL genes by inhibiting miR156, thereby accelerating flower bud differentiation. This offers a reference for regulating flower bud differentiation and blossoming period in O. fragrans through temperature regulation [46].

Similarly, OfCOR27 demonstrates high expression in O. fragrans flower buds, with cultivar- and stage-specific expression patterns. Its role in flowering regulation was verified through overexpression and mutant analyses in A. thaliana, which is consistent with the previous finding that rosette leaf count in A. thaliana shows a positive correlation with flowering time [47]. This suggests that the flower bud phase serves as a key stage in the modulation of O. fragrans’ flowering timing.

The current characterization demonstrates that OfCOR27 expression levels in O. fragrans ‘Sijigui’ were higher during the flower bud sprouting stage compared to ‘Xiaoyesugui’, which is associated with the multiple-flowering trait of ‘Sijigui’. ‘Sijigui’ undergoes multiple rounds of flower bud differentiation throughout the year [48], while ‘Xiaoyesugui’ mainly initiates flower bud differentiation in autumn [49]. We speculate that the high expression of OfCOR27 during the floral bud activation stage in the ‘Sijigui’ cultivar may provide an important molecular basis for its frequent floral bud differentiation cycles. Specifically, it might play a key role in initiating or accelerating each round of floral bud differentiation. This hypothesis requires further validation through temporal expression profiling and functional studies.

3.5. Functional Prospects and Future Research Directions of OfCOR27

In this research, the OfCOR27 gene family in O. fragrans was characterized in multiple dimensions, including characterization of protein physicochemical characteristics, conserved domains, protein structure, and phylogenetic evolution. Expression pattern analysis indicated that OfCOR27 exhibits elevated expression in O. fragrans flower buds and demonstrates cultivar- and stage-specific expression patterns, which are correlated with the flowering time. Overexpression of OfCOR27 upregulated genes promoting flowering and genes related to hormone synthesis. Conversely, the AtCOR27 mutant displayed disordered gene expression, accompanied by postponed bolting and flowering, plus a higher rosette leaf count. These results imply that OfCOR27 likely functions in modulating flowering timing in O. fragrans.

In this research, the focus was solely on the single OfCOR27 gene; the interaction network between OfCOR27 and other genes was not thoroughly explored, and the research was restricted to expression analysis without genetic transformation verification. Based on the association between the high expression of OfCOR27 in the ‘Sijigui’ cultivar and its recurrent flowering trait, future studies could employ genetic transformation in O. fragrans (e.g., overexpression or gene editing) to directly validate its function. Additionally, screening for its interacting proteins using techniques such as yeast two-hybrid and co-immunoprecipitation (Co-IP) will help construct its regulatory network, thereby providing deeper insight into the mechanistic role of OfCOR27 in flowering regulation of O. fragrans. From a long-term perspective, editing the light- and hormone-responsive elements in the OfCOR27 promoter offers a potential theoretical foundation for breeding new O. fragrans varieties adapted to different light and temperature conditions. Furthermore, the key directions of experimental verification in future work should include three aspects. First, explore other phenotypes of transgenic O. fragrans under abiotic stresses (such as drought, low temperature, and salt stress) to clarify whether OfCOR27 is involved in the cross-talk between flowering regulation and abiotic stress response, expanding the understanding of its multi-functional roles. Second, identify its upstream transcription factors using techniques like yeast one-hybrid, which can reveal the regulatory mechanisms initiating OfCOR27 expression and clarify the upstream signaling pathways involved. Third, screen downstream target genes through transcriptome analysis, combine with the existing results of flowering and hormone-related gene expression, and systematically sort out the downstream regulatory cascade of OfCOR27, so as to improve the complete regulatory network of OfCOR27 in O. fragrans flowering regulation. These experimental verifications will further confirm the functional characteristics of OfCOR27 and provide more comprehensive theoretical support for its application in O. fragrans molecular breeding.

4. Materials and Methods

4.1. Materials and Growth Conditions

The plant materials included O. fragrans ‘Sijigui’, O. fragrans ‘Xiaoye Sugui’, N. benthamiana, and wild-type A. thaliana. The O. fragrans cultivars were collected from the central greenbelt of Nanjing Forestry University campus (32°03′ N, 118°48′ E) between May and October 2023. During the sampling period, the average temperature was 18–28 °C, the average sunshine duration was 8–10 h·d−1, and the soil type was neutral loam. Each cultivar was sampled from 3 healthy, uniformly growing adult plants (≥3 years old) to ensure genetic consistency, covering the entire flowering cycle. Three biological replicates were collected per sample; each replicate consisted of flower buds/flowers from one individual plant, and samples from different plants were not mixed. To ensure freshness and biological activity, all collected tissues were immediately flash-frozen in liquid nitrogen after harvesting and stored at –80 °C (Supplementary Materials Figure S1). Wild-type A. thaliana seeds (preserved in our laboratory under dry, low-temperature conditions at 4 °C) were germinated and grown in a controlled growth chamber under the following conditions: a 16 h light/8 h dark photoperiod, light intensity of 120 μmol·m−2·s−1, temperature of 22 ± 1 °C, and relative humidity of 60 ± 5%. For experimental use, seeds were sown with a batch size of n = 12. Seeds of N. benthamiana (preserved in our laboratory and used for subcellular localization) were sown in sterile soil and grown to 3–4-week-old seedlings, with n = 9 plants reserved for experiments. All growth and sampling conditions were strictly controlled and documented to ensure experimental reproducibility.

4.2. Identification of COR27 Gene Family in O. fragrans

The genomic sequence of A. thaliana was acquired from the TAIR database. Employing the COR27 protein sequence of A. thaliana as the query, a BLASTP analysis was performed against the entire O. fragrans genome using TBtools v2.360 [50]. This homology search was intended to identify potential OfCOR27 homologs. The retrieved candidate protein sequences were subsequently subjected to an examination of conserved domains using the Conserved Domain Database (CDD) online tool available at NCBI. Members lacking the characteristic conserved domains were excluded, culminating in a final set of putative OfCOR27 protein sequences exhibiting significant homology.

4.3. Phylogenetic Analysis of OfCOR27 Proteins

The phylogenetic tree was constructed from protein sequences of O. fragrans, A. thaliana, N. tabacum, O. sativa, V. vinifera, and O. europaea by the neighbor-joining method in MEGA11 [51].

4.4. Protein Sequence and Conserved Motif Analysis of the OfCOR27 Gene Family

The protein domain architecture of the OfCOR27 family was analyzed with GSDS 2.0 [52]. Conserved motifs within the OfCOR27 gene family of O. fragrans were identified by MEME [53], with the motif count set to 6 and the motif width allowed to range from 6 to 50 amino acid residues. Sequence alignment of the gene family’s protein sequences was carried out with ESPript 3 [54].

4.5. Prediction and Identification of Cis-Acting Regulatory Elements

Promoter sequences, which are defined as the 2 kb regions upstream of the ATG, were retrieved for all OfCOR27 genes by means of TBtools. These sequences were subjected to a search for cis-acting elements through PlantCARE. Subsequently, the identification, categorization, and visualization of flowering-time-related elements were carried out in TBtools.

4.6. Protein Structural Characterization and Bioinformatics Tools Analysis

Characteristics of the protein encoded by OfCOR27 encompassed an assessment of its physicochemical properties through Expasy’s ProtParam [55] (http://www.expasy.org, accessed on 18 July 2025), a prediction of its secondary structure utilizing SOPMA, and a modeling of its tertiary structure with SWISS-MODEL [56].

4.7. Subcellular Localization Analysis

The coding sequence (CDS) of O. fragrans “Sijigui” served as the basis for the design of gene-specific primers. Employing petal cDNA as the template, the OfCOR27 CDS was amplified, corresponding to the region excluding the stop codon, through polymerase chain reaction (PCR). Subsequently, the Gateway BP recombination reaction was performed to clone the amplified segment into the pMDC43 entry vector. Following construction, the plasmid was used to transform E. coli DH5α competent cells. After verification by sequencing, the confirmed recombinant plasmid was utilized for subcellular localization experiments (Supplementary Materials Figure S2). For transient transformation in N. benthamiana, Agrobacterium tumefaciens GV3101 was cultured in Luria–Bertani broth at 28 °C, 200 rpm, to an OD600 of 0.5–0.6. Bacterial cells were harvested by centrifugation and resuspended in infiltration buffer to a final D600 of 0.8–1.0. The mixture was incubated in darkness for 2–4 h. The bacterial suspension was infiltrated into the abaxial surface of leaves from 3–4-week-old tobacco plants until full infiltration (Supplementary Materials Figure S3). After 2–3 days of cultivation in a growth chamber, fluorescence signals in the abaxial epidermal cells of tobacco leaves were captured using a confocal laser scanning microscope.

4.8. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)

Total ribonucleic acid (RNA) was obtained from the petals of O. fragrans ‘Sijigui’ utilizing the TIANGEN RNAprep Pure Plant Total RNA Extraction Kit (Catalog Number: DP411, TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China). The manufacturer’s instructions were strictly followed when performing the particular extraction processes. The kit employed for complementary deoxyribonucleic acid synthesis was the PrimeScript™ RT Master Mix (Catalog Number: RR036Q), which was procured from Takara Bio Inc., Dalian, China. qRT-PCR was conducted using the SYBR Green Pro Taq HS qRT-PCR Kit (Catalog Number: AG11701) from Nanjing Vazyme Biotech Co., Ltd., Nanjing, China, following the instructions provided with the kit. For every sample, three technical duplicates were created, with OfACT [57] serving as the reference gene. The relative gene expression levels were computed according to the 2−ΔΔCt method. Statistical analysis was executed using the Statistical Package for the Social Sciences. Statistical significance was denoted by a p-value of less than 0.05, and graphs were generated using Origin 2025b.

4.9. Functional Verification of OfCOR27 Overexpression and Mutant Plants

4.9.1. Construction and Identification of Overexpression Vector

The overexpression vector pMDC32-OfCOR27 was constructed as follows: The full-length CDS (without the stop codon) of OfCOR27 was PCR-amplified using specific primers designed based on the SpeI and AscI sites in the multiple cloning site of the pMDC32 vector (preserved in our laboratory, conferring Kanamycin and Hygromycin resistance). The PCR product was then assembled with the linearized pMDC32 vector via homologous recombination. The resulting recombinant vector was introduced into E. coli DH5α competent cells. Positive clones were selected and validated by colony PCR (Supplementary Materials Figure S4) followed by sequencing to confirm the correct insertion and absence of mutations in the OfCOR27 sequence.

4.9.2. Screening and Identification of Transgenic A. thaliana

The sequence-verified recombinant plasmid pMDC32-OfCOR27 was introduced into A. tumefaciens GV3101 competent cells using the freeze–thaw method. Positive clones identified by colony PCR were cultured, and the resulting bacterial suspension was used for A. thaliana transformation. Wild-type A. thaliana Col-0 seeds were surface-sterilized and sown separately on Murashige and Skoog (MS) medium in three biological replicates, with 12 seeds per replicate. Plants were grown until the bolting stage (3–4 weeks old). From each replicate, 8 uniform and healthy plants were selected for floral dip transformation (total: 3 replicates × 8 plants = 24 infected plants), while the remaining 2–3 seedlings per replicate were retained as backups. To improve transformation efficiency, a second round of floral dip infection was performed one week after the first infection.

After infection, plants from each biological replicate were cultivated separately to maturity, and T0 seeds were harvested. The T0 seeds were surface-sterilized and sown independently on MS medium supplemented with 50 mg/L Hygromycin (Hyg) for resistance screening, with 12 seeds sown per biological replicate. This yielded 8–10 healthy seedlings per replicate (actual positive rate approximately 60–80%, consistent with the conventional transformation efficiency of the floral dip method). Green, normally growing T1 putative positive seedlings were selected, while chlorotic or stunted non-transgenic seedlings were discarded. Positive plants were confirmed by PCR using primers specific to the target gene and the vector Nos terminator, followed by 1% agarose gel electrophoresis (Supplementary Materials Figure S5). From each replicate, 3–4 confirmed positive plants with clear target bands were self-pollinated, and their T2 seeds were harvested individually. For homozygous line screening, 20 T2 seeds from each T1 plant were sown on MS medium containing 50 mg/L Hyg. Lines in which 100% of the seedlings exhibited hygromycin resistance were identified as homozygous T2 lines and used for subsequent experiments.

4.9.3. Identification of AtCOR27 Mutants

Seeds of the AtCOR27 mutant were purchased from Fuzhou Airosha Biotechnology Co., Ltd. (Fuzhou, China). The seeds were surface-sterilized and sown on MS medium in three biological replicates, with 12 seeds per replicate, and grown to adulthood. Homozygous mutants were genotyped using a dual-primer approach with the following primer sequences: AtCOR27-LP: 5′-CGTGAATGAATATTTTGACATGG-3′, AtCOR27-RP: 5′-CTTCAATCGCAAAGAAGCAAC-3′, and AtCOR27-LB: 5‘-ATTTTGCCGATTTCGGAAC-3′. PCR products were analyzed by 1% agarose gel electrophoresis to identify homozygous mutant lines. For each biological replicate, total RNA was extracted from the leaves of confirmed homozygous plants. The absence of AtCOR27 transcripts was subsequently verified by semi-quantitative PCR to ensure a loss-of-function phenotype (Supplementary Materials Figure S6).

4.9.4. Comparison of Flowering Phenotypes and Gene Expression Between OfCOR27 Overexpression Lines and AtCOR27 Mutants

Three homozygous OfCOR27 overexpression lines, three homozygous AtCOOR27 mutant lines, and wild-type Arabidopsis Col-0 were selected. For each line, three biological replicates were established, with 10 plants per replicate. All seeds were sown simultaneously on MS medium and grown in a controlled climate chamber under the following conditions: 16 h light/8 h dark photoperiod, light intensity 120 μmol·m−2·s−1, temperature 22 ± 1 °C, and relative humidity 60 ± 5%. After 10 days, uniformly grown seedlings were transplanted into a mixed substrate consisting of nutrient soil:vermiculite = 5:2 and continued to be cultivated under the same conditions. Data were collected independently for each biological replicate. The following phenotypic traits were recorded: bolting time (days required for the bolt to reach 1 cm), flowering time (days from sowing to the opening of the first flower), and rosette leaf number at flowering. Differences between groups were analyzed using independent-sample t-tests, with statistical significance set at *p* < 0.05. Flower tissues were collected from 4-week-old plants in each biological replicate. Total RNA was extracted following the protocol described in Section 4.8, and qRT-PCR was performed to quantify the expression levels of flowering-related genes and hormone-metabolism-related genes. Each qRT-PCR was run with three technical replicates. Relative expression levels were calculated using the 2−ΔΔCt method, and statistical analysis was conducted by integrating data from the three biological replicates (*p* < 0.05).

5. Conclusions

This study clarifies that OfCOR27 is a positive regulator of plant flowering through phenotypic analysis of A. thaliana overexpressing OfCOR27, AtCOR27 mutants, and wild-type plants. The homozygous OfCOR27 overexpression lines exhibit earlier bolting, earlier flowering, and a reduced number of rosette leaves at flowering, while AtCOR27 mutants show delayed flowering, confirming the evolutionary conservation of COR27 homologous genes in flowering regulation. Combined with the high expression of OfCOR27 during the bud sprouting stage in O. fragrans ‘Sijigui’, OfCOR27 may not only accelerate flowering but also regulate flowering frequency in woody plants. At the molecular level, OfCOR27 overexpression may promote reproductive development in A. thaliana by enhancing the expression of flowering-promoting genes and altering hormone levels. This study provides molecular evidence for the function of OfCOR27 in plant growth and development, offers a feasible strategy for improving plant flowering time through genetic engineering, and lays a theoretical foundation for deciphering OfCOR27-mediated flowering regulation in O. fragrans, thereby supporting the genetic improvement of flowering traits in woody ornamental plants.

Acknowledgments

We sincerely thank the dedicated guidance provided by our supervisors and Shenglian Li throughout the research and the writing of this paper. Appreciation is extended to the editorial team and anonymous reviewers whose constructive comments significantly strengthened the manuscript.

Abbreviations

The following abbreviations are used in this manuscript:

COR27 COLD-REGULATED 27
AtLFY Arabidopsis thaliana LEAFY
AtFT Arabidopsis thaliana FLOWERING LOCUS T
AtAP1 Arabidopsis thaliana APETALA1
AtFUL Arabidopsis thaliana FRUITFULL
AtPHYB Arabidopsis thaliana PHYTOCHROME B
AtIAR3 Arabidopsis thaliana IAA-ALANINE RESISTANT 3
AtACS5 Arabidopsis thaliana 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE 5
AtILR1 Arabidopsis thaliana IAA-LEUCINE RESISTANT 1
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Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15040610/s1, Figure S1: O. fragrans phenotypes in different periods (The flower bud scale is 1 mm, and the rest are 1 cm). A, O. fragrans ‘Siji’ Flower bud differentiation stage; B, O. fragrans ‘Siji’Calyx emergence stage; C, O. fragrans ‘Siji’ Ballshape stage; D, O. fragrans ‘Siji’ Budding stage; E, O. fragrans ‘Siji’ Primary blooming stage; F, O. fragrans ‘Siji’ Full blooming stage; G, O. fragrans ‘Siji’ Late full stage; H, O. fragrans ‘Xiaoye Sugui’ Flower bud differentiation stage; I, O. fragrans ‘Xiaoye Sugui’ Calyxemergence stage; J, O. fragrans ‘Xiaoye Sugui’ Ball-shape stage; K, O. fragrans ‘Xiaoye Sugui’ Budding stage; L, O. fragrans ‘Xiaoye Sugui’ Primary blooming stage; M, O. fragrans ‘Xiaoye Sugui’ Full blooming stage; N, O. fragrans ‘Xiaoye Sugui’ Late full stage; Figure S2: Cloning and analysis of OfCOR27 from O. fragrans. (a) Results of total RNA electrophoresis in O. fragrans. M, 2000DNA Marker; 1–2, Total RNA. (b) PCR amplification products of the O. fragrans. 1–2, OfCOR27 gene; Figure S3: Subcellular localization vector construction glue diagram. (a), Detection of E. coli liquid by PCR; (b), Agrobacterium tumefaciens PCR assay; (c), pMDC43-GFPAgrobacterium tumefaciens PCR assay. M, 2000DNA Marker; A-1~A-8, OfCOR27gene; B-1~B-8, OfCOR27gene.; Figure S4: Colony PCR Detection of Overexpression Vector in E. coli DH5α and Agrobacterium tumefaciens. (a) Detection of Overexpression Vector in E. coli DH5α by Colony PCR. (b) Detection of A. tumefaciens by Colony PCR. M, 2000 DNA Marker; 1-8, Single colony samples.; Figure S5: Selection and verification of OfCOR27 transgenic Arabidopsis thaliana. (a) Screening of transgenic A. thaliana on selective medium. Left, Transgenic positive seedlings; Right, Non-transgenic seedlings. (b) PCR identification of transgenic A. thaliana. M, 2000 DNA Marker; 1-11, OfCOR27 transgenic lines; 12, Negative control.; Figure S6: Identification and Verification of AtCOR27 Insertional Mutants. (a) PCR identification of AtCOR27 insertional mutants. (b) Semi-quantitative RT-PCR verification of AtCOR27 insertional mutants. (c) Reference gene validation of AtCOR27 insertional mutants.

plants-15-00610-s001.zip (564KB, zip)

Author Contributions

Writing—original draft, J.L.; Data curation, R.C.; Software—Image processing, R.C., J.L., S.L. and D.Z.; Review—editing, J.L., R.C., M.Z. and Y.D. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data are contained within the NCBI.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

plants-15-00610-s001.zip (564KB, zip)

Data Availability Statement

Data are contained within the NCBI.

Articles from Plants are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

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