Genome-Wide Identification and Functional Characterization of TIFY Gene Family in Verbena bonariensis with Insights into VbTIFY16’s Role in Petal

1 Introduction

As a perennial herbaceous species of the genus Verbena in the family Verbenaceae, Verbena bonariensis is recognized as one of the typical and representative perennial herbaceous germplasm resources within this genus [1], has a typical quadrangular stem and grows to a height of 1–1.5 m, belonging to the medium to tall ornamental herb category. The flowers are terminal or axillary, displaying purple-red or blue coloration. Native to South America, it is now widely cultivated in many provinces across China, including the east, south, northwest and southwest. Verbena bonariensis prefers full sunlight and has weak cold resistance. It thrives best at temperatures between 20–30°C, with a flowering period that extends from spring to autumn, offering a long ornamental period [2]. As a flowering plant, it also contributes to insect pollination and is considered a valuable nectar source. In addition to its ornamental value, V. bonariensis is recognized for its medicinal properties, exhibiting efficacy in detoxification, reducing swelling, and relieving spasms. It may serve as an effective hepatoprotective agent and has potential for alleviating liver diseases [3]. Given the escalating demand for V. bonariensis, investigations into its growth, development and stress regulation are of paramount importance.

A transcription factor gene family unique to plants, the TIFY family is distinguished by the presence of a conserved TIFY domain that acts as its representative structural characteristic [4]. According to their conserved sequence characteristics, the TIFY transcription factor family can be divided into four distinct subfamilies, namely TIFY, JAZ, ZML and PPD [5]. All members of this family possess a signature TIFY domain, which harbors the highly conserved core motif TIF [F/Y]XG [6]. The TIFY subfamily members contain only the TIFY domain. The JAZ subfamily possesses a Jas domain that exhibits high sequence homology with the N-terminal sequence of the CCT domain [6,7]. For the ZML subfamily, members carry both a CCT domain and a GATA-type zinc finger domain. In contrast, the PPD subfamily is distinguished by the presence of a PPD domain and a truncated Jas domain [4,5,6].

To date, TIFY genes have been identified in multiple plant species, such as maize with 47 members [8] and Camellia sinensis with 16 [9]. As an important regulatory gene family in plants, members of the TIFY family are involved in the regulation of plant developmental processes, enhancement of stress resistance, and responses to abiotic stress through multiple pathways, and simultaneously perform core regulatory functions in the transduction pathways of plant hormone signals [10].

The TIFY family members are broadly engaged in regulating the development of diverse organs and tissues throughout plant growth. For example, AtTIFY1 (ZML), the first TIFY member identified in A. thaliana, is not only associated with inflorescence development and flowering but promotes petiole and hypocotyl elongation by mediating cell extension as well [11]. Hakata et al. found that OsTIFY11 and OsTIFY10b can increase crop yield by enhancing grain size and grain number, respectively; overexpression of OsTIFY11 also reduces spikelet fertility through JA-mediated regulation, thereby increasing rice yield [12]. Yu et al. demonstrated that SlTIFY2 in tomato promotes lateral bud germination and early flowering, accelerating the transition from vegetative to reproductive growth [13]. In chrysanthemum, In chrysanthemum plants, the regulation of petal size by CmJAZ1 is achieved through protein-protein interaction with bHLH transcription factor CmBPE2. This binding process impedes the transcriptional activation of expansin gene CmEXPA7 by CmBPE2, and since CmEXPA7 acts as a critical gene governing cell expansion, petal growth is consequently restrained [14].

TIFY proteins play a crucial role in response to abiotic stresses. For example, TdTIFY11a enhances the germination rate of wheat under salt stress [9]. ZmTIFY16 promotes root growth and development in both Arabidopsis and maize, improving drought and salt resistance [15]. AtJAZ10 in Arabidopsis [16] and OsJAZ8 in rice [17] contribute to enhanced disease resistance in plants. TaJAZ1 increases resistance to biotic stress in wheat [18]. And overexpression of CaTIFY7 and CaTIFY10b confers enhanced cold tolerance in pepper [19].

While TIFY transcription factors act as key regulators governing plant growth and development, research on the identification and expression characteristics of the TIFY gene family in V. bonariensis remains extremely limited in the academic community. Within this research, we performed genome-wide identification of TIFY gene family members in V. bonariensis using genomic data obtained by our research team. Physicochemical properties, gene structures, domains, conserved motifs, phylogenetic relationships, chromosomal distribution and cis-acting elements of these genes were analyzed, and their expression patterns in various tissues were further examined via a combination of transcriptome sequencing and qRT-PCR. Furthermore, subcellular localization analysis of the VbTIFY16 gene belonging to the TIFY family verified its nuclear localization. An overexpression vector of this gene was constructed and stably transformed into tobacco, demonstrating that VbTIFY16 negatively regulates petal size in transgenic plants by inhibiting cell expansion. This study lay a theoretical foundation for elucidating the regulatory mechanism of petal size in V. bonariensis and provide additional candidate genes for molecular breeding.

2 Materials and Methods

2.1 Plant Materials

Verbena bonariensis seeds were obtained from Benary Seed Company (Hann. Münden, Germany). Seedling maintenance and multiplication were carried out in the Germplasm Resource Nursery at the Key Laboratory of Mountain Plant Resources Protection and Germplasm Innovation (Ministry of Education), Guizhou University. Wild-type tobacco seeds were provided by our laboratory’s nursery, and all plant materials were identified and authenticated by Dr. Yan Li and Dr. Sumeera Asghar.

2.2 Identification of TIFY Gene Family Members in V. bonariensis

The V. bonariensis genome annotation (PRJCA027875) was downloaded from the National Genomics Data Cen ter (NGDC, https://ngdc.cncb.ac.cn) [20], downloaded the TIFY conserved domain (PF06200) from Pfam database (http://pfam.xfam.org/). Candidate members containing the conserved structural domain were obtained by aligning the conserved structural domain to the genomic data of V. bonariensis using HMMER 3.1 (http://hmmer.org/download.html). In addition, 18 Arabidopsis TIFY members were compared to the Verbena willow genome using BLASTP (E-value < 1 × 10−5) to obtain candidate members. All the candidate members obtained were submitted to: SMART (http://smart.embl-heidelberg.de/) and NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to determine the conserved structural domains of TIFY, and finally obtain the TIFY family members.

2.3 Physico-Chemical Properties of TIFY Gene Family in V. bonariensis

The physicochemical parameters of V. bonariensis TIFY family proteins, including protein length, molecular weight, theoretical isoelectric point (pI), instability index, and grand average of hydropathicity (GRAVY), were analyzed using the ProtParam (https://web.expasy.org/compute_pi/) [21]. The secondary structure composition was analyzed using the SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html). Transmembrane helices were predicted via the TMHMM (https://services.healthtech.dtu.dk/services/TMHMM-2.0/), subcellular localization was determined by WoLF PSORT (https://wolfpsort.hgc.jp/) [22]. And the precise positions of conserved structural domains were mapped using NCBI’s Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

2.4 Visualization of Gene Structures, Domains, and Conserved Motifs in the TIFY Gene Family of V. bonariensis

The intron-exon organization profiles of all identified VbTIFY genes were retrieved from the official genome annotation dataset of V. bonariensis. Conserved domain information was acquired by searching against the NCBI CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). A total of 10 evolutionarily conserved motifs were identified using the MEME (http://meme-suite.org/tools/meme) [23] with default parameter configurations. All the above structural features were ultimately visualized using TBtools software (v2.042) [24].

2.5 Analysis of Phylogenetic Evolution and Chromosomal Distribution Characteristics of TIFY Genes in V. bonariensis.

Using the MUSCLE program within MEGA software, multiple sequence alignment was performed between the identified TIFY family genes from V. bonariensis and from A. thaliana. A phylogenetic tree was generated using MEGA version7.0 Bootstrap with parameter 1000 [25]; Subsequently the phylogenetic tree of VbTIFY genes was then reconstructed via the neighbour-joining method. Finally, the constructed phylogenetic tree was annotated and visualized using the Evolview v2 (https://evolgenius.info/evolview-v2/) [26]. Based on the genomic annotation data of V. bonariensis, the chromosomal localization of members of the VbTIFY gene family was visualized using TBtools v2.042 [24].

2.6 Cis-Acting Regulatory Element Analysis of TIFY Gene Family Members in V. bonariensis

The 2000 bp genomic sequences upstream of the translation initiation codon (ATG) of each identified VbTIFY gene were extracted from the V. bonariensis genome file using TBtools v2.042 [24]. Putative cis-acting regulatory elements embedded in these promoter sequences were annotated via the PlantCARE online database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [27]. The distribution patterns and functional classification of all identified cis-elements were subsequently visualized and plotted using the same TBtools software [24].

2.7 Characterization of TIFY Gene Family Collinearity in V. bonariensis

Genome-wide datasets and annotation files were acquired on the basis of V. bonariensis genome annotation. Collinear relationships among the gene family members were analyzed using the MCScanX algorithm [28], and TBtools v2.042 was subsequently applied for figure visualization [24].

2.8 Characterization of Expression Discrepancies in TIFY Gene Family

Using the transcriptome dataset generated from V. bonariensis, we retrieved the expression level (FPKM) values of all identified VbTIFY genes across three distinct tissues (flowers, stems, and leaves) and in samples collected pre- and post-12-hour 4℃ cold stress. To eliminate the influence of extreme values, we normalized the raw expression data via log2(FPKM + 1) conversion, and subsequently generated heatmaps to visualize the expression profiles using TBtools 2.042 [24].

2.9 qRT-PCR Verification

Two-month-old healthy Verbena bonariensis plants were selected as experimental materials. Different organs (leaf, flower, and stem) were sequentially collected, placed into sterile, nuclease-free cryotubes, and immediately frozen in liquid nitrogen. These samples were then stored at −80°C for subsequent RNA extraction. Total RNA was isolated using the OMEGA Extraction Kit (OMEGA, Norwalk, CT, USA), and first-strand cDNA was synthesized via reverse transcription using the StarScript III RT Kit. Quantitative real-time PCR (qRT-PCR) reactions were performed on a qTOWER3G Real-Time PCR System (Analytik Jena, Beijing, China) using 2× RealStar Fast SYBR qPCR Mix (GENStar, Beijing, China), with the VbACTIN gene serving as the internal reference. The 10 μL reaction system consisted of 1 μL of cDNA template, 0.5 μL of reverse primer, 0.5 μL of forward primer, 5 μL of 2× RealStar Fast SYBR qPCR Mix, and DEPC-treated water to a final volume of 10 μL. Three biological replicates and three technical replicates were set up for each sample. Additionally, primers were designed using NCBI Primers—blast and Primer Premier 5 software. The relative expression level was calculated using the 2−ΔΔCT method [29], and results are expressed as mean ± standard deviation.

2.10 Subcellular Localization of VbTIFY16

The pCAMBIA1300-VbTIFY16-EGFP fusion expression vector was constructed using the seamless cloning method, and transformed into Nicotiana benthamiana leaves via Agrobacterium tumefaciens strain GV1301-mediated transient transformation. After 2 d of dark incubation, the subcellular localization of VbTIFY16 protein in tobacco leaf epidermal cells was observed using a laser scanning confocal microscope.

2.11 Plasmid Construction and Plant Transformation

The complete coding sequence of VbTIFY16 was amplified via PCR and cloned into the pCAMBIA1301-35S vector using double restriction enzyme digestion, generating the overexpression construct pCAMBIA1301-35S-VbTIFY16. The constructed vector was transferred into Agrobacterium tumefaciens strain EHA105, followed by Agrobacterium-mediated transformation of Nicotiana benthamiana referring to the protocol described by Ning et al. [30]. As reported by Jefferson et al. [31], histochemical staining of β-glucuronidase (GUS) was employed to validate the identity of transgenic plants.

2.12 Phenotypic Analysis of Transgenic N. benthamiana under Normal Conditions

Both transgenic overexpression (OE) plants and wild-type (WT) control plants were maintained in a greenhouse culture system for comprehensive phenotypic characterization. At 65 days following transplantation, multiple growth-related phenotypic indices (plant height, leaf length, leaf width and leaf area) were collected and statistically analyzed; the floral traits (flower diameter and petal area) were assayed at the full blooming (FB) developmental stage. To characterize the cellular features of petals, petal tissue samples were harvested from OE and WT for sectioning, which was followed by morphological observation and size measurement of petal cells. The morphology of petal epidermal cells was observed and photographed using a microscope (Olympus, BX43, Tokyo, Japan) [32]. Cell length and area were determined using ImageJ software (NIH, Bethesda, MD, USA), and the total number of epidermal cells in petals was calculated by multiplying the average number of cells per 1 mm2 by the total petal area.

3 Results

3.1 Identification and Physicochemical Property Analysis of VbTIFY

After comprehensive screening and identification, 16 VbTIFY genes were identified as from VbTIFY1 to VbTIFY16 (Table 1). The encoded proteins had amino acid lengths of 136–372 molecular weights of 15,075.11–40,696.34 Da, and theoretical pI values from 4.74 to 9.75. Thirteen out of 16 VbTIFYs had values higher than 7.0, indicating that most of the V. bonariensis TIFYs were alkaline proteins. Protein instability coefficients of VbTIFYs proteins ranged from 41.55 (VbTIFY15) to 78.85 (VbTIFY3), and the instability index was noticed higher than 40, which was a variable protein. The aliphatic index varied from 53.7 (VbTIFY6) to 82.76 (VbTIFY13). The overall mean of hydrophobicity ranged from −0.842 (VbTIFY2) to −0.007 (VbTIFY9), The average value of grand average of hydropathicity. Additionally, subcellular localization analysis suggested that all VbTIFY proteins are localized to the nucleus (Table 1). Among these, the nuclear localization of VbTIFY16 was further validated experimentally (see Section 3.8, Fig. 1).

Transmembrane domain analysis confirmed that among the 16 members of the VbTIFY family only VbTIFY9 and VbTIFY12 possessed a single transmembrane domain, while the remaining 15 members exhibited no transmembrane domain characteristics. Secondary structure predictions showed that all VbTIFY members had no β bridges, among which random coil was the most common secondary structure in the amino acid sequence of V. bonariensis TIFY, accounting for 70.81% (VbTIFY13) to 85.95% (VbTIFY9), followed by α helix 8.75% to 21.08%, and extended chain 4.21% to 10.29% (Table 2).

Figure 1: Subcellular localization of VbTIFY16.

3.2 Structural Composition and Conservative Motif Analysis of TIFY Family Proteins in V. bonariensis

The results revealed the presence of four putative conserved domains within the VbTIFY family proteins: the TIFY domain, JAS motif domain, CCT domain, and GATA domain. All 16 VbTIFY proteins contain the TIFY domain, confirming their classification as members of the TIFY family. The CCT and GATA domains were exclusively identified in VbTIFY10/11/15, indicating that these three proteins are classified into the ZML subfamily. VbTIFY06 contains only the TIFY domain, suggesting possible structural loss during evolution. The remaining 12 members all possess two conserved domains: TIFY and JAS (Fig. 2). A total of 10 conserved motifs were identified among the TIFY family members in V. bonariensis (Fig. 3). The number of conserved motifs varied among different VbTIFY proteins, ranging from 1 to 6. All VbTIFY members shared Motif 1, which corresponds to the TIFY domain (Fig. 2). The findings confirmed that the VbTIFY gene family has retained high evolutionary conservation. Further phylogenetic analysis indicated that proteins within a given clade displayed analogous domain organizations and conserved motif patterns, which suggests that genes with comparable motif compositions are likely to have related biological functions. Furthermore, Exon-intron structures and 5′/3′ UTR sequences of the 16 identified VbTIFY genes in V. bonariensis were systematically analyzed, which provides essential support for clarifying the structural heterogeneity of the VbTIFY gene family. The findings indicated that the number of exons varied significantly among different members, ranging from 2 to 10. Except for VbTIFY06, which contained 3 non-coding regions, the number of non-coding regions in other VbTIFY members ranged from 0 to 2, indicating that the VbTIFY family genes are relatively conserved during evolution.

Figure 2: Conserved motif profiles, gene structural characteristics and evolutionary relationships of the TIFY gene family in V. bonariensis.

Figure 3: Conservative motif of TIFY gene family members in V. bonariensis.

3.3 Phylogenetic Tree and Chromosome Mapping of VbTIFY

To elucidate the evolutionary divergence of TIFY proteins in V. bonariensis, multiple sequence alignment was performed on 34 TIFY protein sequences, (16 from V. bonariensis and 18 from A. thaliana), followed by the construction of a phylogenetic tree using the neighbor-joining (NJ) method. (Fig. 4). Phylogenetic analysis results showed that the 34 TIFYs were divided into six independent groups, which were sequentially named Group 1 to Group 6. Members of the VbTIFYs family were mainly concentrated in Groups 1, 3, 5 and 6, while only one VbTIFY family member was contained in each of Group 2 and Group 4. Based on the structural characteristics of the TIFY domain, this gene family can be divided into four major subfamilies: JAZ, ZML, TIFY and PPD. Group 1 belongs to the ZML subfamily, containing three members including VbTIFY10/11/15, as well as three members derived from A. thaliana. Group 2 falls into the TIFY sub-family and contains one member, VbTIFY6. Group 4 is assigned to the PPD sub-family and includes one member, VbTIFY4. The remaining three groups Group 3/5/6 belong to the JAZ subfamily and contain a total of 11 members. Furthermore, the phylogenetic tree analysis demonstrated tight clustering of TIFY proteins within the same group, a pattern consistent with the tree constructed using VbTIFY proteins in V. bonariensis. (Fig. 2 and Fig. 4).

Figure 4: Phylogenetic tree of TIFY gene family members from V. bonariensis and A. thaliana.

The tree includes 16 VbTIFY proteins (red ★) and 18 AtTIFY proteins (black ●). The proteins are divided into six subgroups (Groups 1–6), corresponding to four subfamilies: ZML (Group 1), TIFY (Group 2), JAZ (Groups 1). Actually (Groups 3,5,6), and PPD (Group 4). The NJ tree was generated with 1000 bootstrap replicates.

16 VbTIFY genes were mapped to the chromosomes of V. bonariensis (Fig. 5). It is important to note that V. bonariensis is an autotetraploid species (4n), and the genome assembly used in this study represents a single subgenome (n). Therefore, the 16 VbTIFY genes identified here are expected to represent only a subset of the total TIFY complement, with additional copies potentially present in the three remaining subgenomes. All members were successfully located, with the distribution across chromosomes as follows: Chr.02 and Chr.06 contained the highest number of VbTIFY genes, each harboring four members, followed by Chr.05 with three members. Chr.03 and Chr.07 each carried two VbTIFY genes, while Chr.04 contained only one.

Figure 5: Chromosomal location of TIFY gene family members in V. bonariensis.

3.4 Colinearity Analysis of TIFY Gene Family Members in V. bonariensis

To investigate the evolutionary conservation of the TIFY gene family, collinearity analysis detected four homologous gene pairs (Red line in Fig. 6). As tabulated in Table 3, Ka/Ks ratios for these 4 pairs were all <1, demonstrating that the homologous genes evolved under negative selection (purifying selection pressure).

Figure 6: Colinearity relationship of TIFY gene family members in V. bonariensis.

3.5 Analysis of Cis-Elements in the Promoter Regions of VbTIFY Genes

To elucidate the putative regulatory roles of the TIFY gene family in V. bonariensis, we screened the 2000 bp promoter regions upstream of the start codon for cis-acting regulatory elements (Fig. 7). The results revealed that VbTIFY genes harbor a diverse array of cis-elements, with light-responsive elements being the most prevalent (93), abscisic acid responsiveness (64), MeJA-responsiveness (28), anaerobic induction (35), low-temperature-responsive elements (10), auxin responsiveness (7), gibberellin responsiveness (8), among others. Specifically, 14 members contained abscisic acid-responsive cis-elements, apart from VbTIFY6 and VbTIFY13. MeJA-responsive elements were identified in eight VbTIFY genes (VbTIFY2/3/4/8/10/11/15/16). Regarding other phytohormone-related cis-elements, for instance, VbTIFY8 contained five types of auxin, gibberellin, salicylic acid, abscisic acid, and MeJA responsiveness. The presence of these cis-regulatory elements indicates that the TIFY gene may play a role in the cross-regulation of multiple hormones.

Regarding environmental response elements, all members carried multiple light-responsive cis-elements. Thirteen genes contained cis-elements essential for anaerobic induction, excluding VbTIFY6, VbTIFY11, and VbTIFY13. Additionally, six genes (VbTIFY3/4/5/6/12/13) contained defense and stress responsiveness elements; seven (VbTIFY2/3/5/9/10/13/15) contained low-temperature responsiveness elements; and nine (VbTIFY2/3/4/6/7/11/13/14/15) contained drought-inducibility elements. Furthermore, several genes carried tissue-specific or functionally specific cis-elements. For example, VbTIFY4/6/10/15 contained elements associated with endosperm expression; VbTIFY4/6/7/10/11 possessed elements related to meristem expression; and VbTIFY8 contained a cis-acting element involved in seed-specific regulation (Fig. 7).

Figure 7: Cis-elements analysis in the promoters of TIFY genes in V. bonariensis. The 2000 bp promoter sequences of VbTIFY genes were used to analyze cis-elements.

3.6 Differences in the Expression of TIFY Gene Family Members in V. bonariensis

To further investigate the regulatory roles of TIFY gene family members in the growth and development of V. bonariensis, we calculated and visualized the transcriptomic FPKM values of 16 TIFY family members across three different tissues of V. bonariensis (Fig. 8 and Fig. 9). The results demonstrated that VbTIFY genes exhibited distinct expression profiles across different tissues of V. bonariensis. Specifically, 13 VbTIFY genes were expressed in all tissues, while three genes showed no or very low expression in most tissues. VbTIFY07/01/08/03 were highly expressed in leaves; VbTIFY05/09/11 exhibited higher expression in stems. Notably, VbTIFY16 exhibited a marked upregulation in floral tissues relative to other plant organs, implying a potential role in flower development and function. Analysis of the expression patterns of TIFY gene family members in V. bonariensis under cold stress indicated that the expression levels of 12 members were significantly elevated after low-temperature treatment. Among them, two members (VbTIFY14/15) reached peak expression at 3 h; six members (VbTIFY01/02/05/08/12/13) peaked at 8 h; two members (VbTIFY03/09) peaked at 12 h; and two members (VbTIFY07/16) peaked at 24 h. In contrast, four members showed decreased expression levels. A series of abiotic stress-responsive cis-acting elements were detected in the promoter regions of 12 upregulated VbTIFY genes, which suggests that these members may play potential regulatory roles in plant responses to cold stress and the development of cold tolerance. Collectively, our results lay a solid theoretical foundation for subsequent research on deciphering the cold stress response mechanism in V. bonariensis.

Figure 8: Tissue-specific expression of TIFY genes in V. bonariensis.

Figure 9: Expression heatmap of Cold stress responsive expression in V. bonariensis.

3.7 qPCR-Based Characterization of VbTIFY Gene Expression in Flower Stem and Leaf

To validate the RNA-seq results from different tissues of V. bonariensis, qRT-PCR analysis on six (VbTIFY01, VbTIFY03, VbTIFY05, VbTIFY09, VbTIFY12, and VbTIFY16) randomly selected genes from the RNA-seq dataset (Fig. 10) results demonstrated expression profiles consistent with RNA-seq analysis (see Fig. 8). Notably, VbTIFY16 showed significant up-regulation in flowers, where its expression level was the highest among all the genes analyzed. Our findings indicated that VbTIFY16 may play a critical regulatory part in the growth and development processes of V. bonariensis flowers.

3.8 Subcellular Localization of VbTIFY16 Protein

To investigate the subcellular localization of VbTIFY16, the empty vector pCAMBIA1300-EGFP and recombinant vector pCAMBIA1300-VbTIFY16-EGFP were transformed separately. After 2-3 days of low-light incubation, the subcellular distribution of the Protein-GFP fusion protein in the agroinfiltrated leaves was observed under a laser scanning confocal microscope (LSCM) with excitation at 488 nm. The results showed that strong green fluorescent signals of VbTIFY16-EGFP were detected in the nucleus (Fig. 1), indicating that VbTIFY16 is localized to the nucleus.

Figure 10: Expression profiling of six VbTIFY genes in flowers, stems, and leaves of V. bonariensis. Data are presented as mean ± SD, with three biological replicates (independent individuals) and three technical replicates performed in qRT-PCR assays. Distinct lowercase letters denote significant differences as determined by the LSD test (α = 0.05).

3.9 Overexpression of VbTIFY16 Negatively Regulates Petal Size in Transgenic Nicotiana Benthamiana

To further elucidate the function of VbTIFY16, the 35S: VbTIFY16-GUS construct was introduced into tobacco, leading to the generation of three independent transgenic lines. Observation results showed that at the full petal expansion stage, the VbTIFY16 OE plants exhibited a significantly reduced inflorescence diameter and shorter petals, with their average petal length being 20.3% lower than that of the wild-type (WT) (Fig. 11C,F,G).

To explore the reasons for shorter petals, sections of petals from both transgenic plants and WT plants were prepared following the method described by Ren, and cytological observations were carried out [32] (Fig. 11D). Morphological observation and quantitative measurement of petal epidermal cells revealed that the cell diameter and cell area of VbTIFY16-OE petals were reduced by 20.54% and 40.19% compared with those of WT petals, respectively (Fig. 11I,J). Meanwhile, the total number of epidermal cells in petals showed no significant difference (Fig. 11H). Therefore, we infer that the inhibition of petal growth in VbTIFY16-OE plants is mainly attributed to the impaired cell expansion. In addition, the overexpression of VbTIFY16 transcription factor significantly affected plant growth potential at the vegetative growth stage. Compared with WT plants, VbTIFY16-OE plants exhibited a shorter height phenotype with smaller leaves (Fig. 11). Further statistical analysis confirmed that the plant height, leaf length and leaf width of the overexpression plants were all significantly decreased (Fig. 11 and Fig. 12).

Figure 11: Phenotypic characterization of VbTIFY16 overexpressing transgenic Nicotiana Benthamiana. (A) Gus validation of transgenic tobacco (Bar:1 cm) (B) Phenotype of WTand VbTIFY16-OE transgenic plant in a greenhouse, Bar: 3 cm. (C) Petal Comparison Between WT and VbTIFY16-OE Plants. Bars: 1 cm. (D) The cells in WT and VbTIFY16-OE transgenic plants observed using optical microscope (40×). Bars: 50 μm. (E) WT and VbTIFY16-OE plant heights were measured at 65 days post-transplant. (F,G) WT and VbTIFY16-OE petal length and area: measured at the FB stage. (H,I) Cell length (H) and area (I) analysis in WT and VbTIFY16-OE petal regions at FB stage. (J) Statistical analysis of total number of petal cell in WT and VbTIFY16-OE plant. Three independent transgenic lines (OE-1, OE-2, OE-3) were obtained; representative images of each line are shown in (B–D). (E–J) Data are means of all three independent lines. (n ≥ 9 plants per line, mean ± SD). All significant differences were determined by Student’s t test (ns: not significant (p ≥ 0.05), **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 12: Plant architecture phenotype of VbTIFY16 overexpression transgenic plants at the vegetative stage. (A) Leaf Phenotypes of WT and VbTIFY16-OE Transgenic Plants Bars: 1 cm. (B) WT and VbTIFY16-OE leaf length and width were measured. Data are means of all three independent lines (n ≥ 9 plants per line, mean ± SD). Significant differences were determined by Student’s t test (ns: not significant (p ≥ 0.05), **p < 0.01, ***p < 0.001, ****p < 0.0001).

4 Discussion

The TIFY gene family members (particularly JAZ proteins) represent a class of core regulators induced by jasmonic acid in plants. They typically form regulatory complexes with other proteins or transcription factors, playing pivotal roles in plant growth and development, metabolic synthesis, and responses to environmental stresses [33]. As scientific research continues to advance, the practical application scope of the TIFY gene family is anticipated to broaden considerably. As genome sequencing projects for various species reach completion, comprehensive identification of the TIFY gene family has been achieved in numerous plant lineages, including 18 in the A. thalian [4] and 21 in Actinidia chinensis [34]. This study identified 16 VbTIFY members from V. bonariensis, revealing significant interspecific divergence in the composition of the TIFY gene family. Notably, research on the TIFY gene family in the V. bonariensis genome remains largely unexplored, and their functional roles have not yet been elucidated. Therefore, systematic analysis of the VbTIFY gene family will advance our understanding of the specific biological functions of these genes.

The coding sequence lengths of the 16 identified TIFY proteins showed substantial variation, spanning from 136–372 bp. Their relative molecular masses fell within the range of 15,075.11–40,696.34 Da, while the theoretical isoelectric points (pI) of these proteins varied between 4.74–9.75. All members of the VbTIFY family were categorized as hydrophilic proteins, a finding that aligns with previous reports on Lycium ruthenicum [35]. Furthermore, they were predominantly localized in the nucleus, a result consistent with that observed in birch [36]. Collectively, these data imply that VbTIFY proteins may exert their biological functions mainly through transcriptional regulation in the nucleus.

Based on the constructed phylogenetic tree, the majority of VbTIFY genes clustered in a specific clade displayed consistent exon-intron structural patterns and conserved motif profiles, even though the lengths of some internal introns differed considerably. This phenomenon indicates that the intron organization of homologous genes tends to be evolutionarily conserved—and notably, these structural differences also provide a basis for TIFY family classification while implying potential functional divergence among its members.

In addition to structural observation of the phylogenetic tree, a joint phylogenetic tree of V. bonariensis and A. thaliana was constructed further. All TIFY genes were classified into six groups: Group I contained AtTIFY1/2A/2B and VbTIFY10/11/15, belonging to the ZML subfamily with potential roles in hypocotyl and petiole elongation [37]. Group 2 comprises only AtTIFY8 and VbTIFY6, which may act as repressors of leaf senescence [38]. Group 3 includes AtTIFY6A/6B/7 and VbTIFY7/9/14, while Group 4 contains AtTIFY4A/4B and VbTIFY4, members of the PPD subfamily that regulate lamina size and curvature development [39]. Groups 5 and 6 encompass AtTIFY3B/5A/5B/8A/10A/10B/11A/11B and VbTIFY2/3/5/8/12/13/16; studies on these genes suggest their potential roles in plant defense, growth, and development [40]. Most importantly, the phylogenetic tree analysis provides valuable insight into the functional characterization of different VbTIFY genes.

As key regulatory elements governing gene transcription and expression, cis-acting elements are commonly used to predict the potential functions of gene [41]. In this study, a variety of cis-regulatory elements were identified in the promoter regions of VbTIFY genes, suggesting that these genes have the activation potential to respond to diverse stress signals. For instance, numerous light-responsive cis-acting elements were identified in the promoters of all members of this gene family, suggesting that the expression of VbTIFY genes is modulated by light signals and thus involved in regulating the growth and developmental processes of V. bonariensis.

Notably, most VbTIFY genes contain both stress- and hormone-related regulatory elements. Cis-element analysis implies that these genes not only help V. bonariensis tolerate diverse stresses, but also function as central regulators in plant growth. Existing research indicates that multiple TIFY family genes can be induced by abiotic stresses. For example, OsJAZ genes are responsive to various abiotic stresses such as drought, cold and salinity, whereas OsTIFY11a improves the stress resistance of rice [42]. GsTIFY10a is likely to exert a positive function in mediating plant salt tolerance [43]. This study identified TIFY genes involved in the low-temperature response in V. bonariensis, with 12 TIFY genes being upregulated. These findings suggest that these genes may play an active regulatory role in the abiotic stress response of V. bonariensis, providing important insights for the subsequent identification of resistance genes in this species.

Members of the TIFY family are involved in the development of plant floral organs. For instance, the PnFL-2 protein of morning glory (Pharbitis nil) possesses the conserved TIFY and CCT domains typical of the TIFY gene family. Research findings have shown that constitutive overexpression of PnFL-2 in transgenic plants results in a subtle advancement of flowering time under long-day photoperiods, as compared to their wild-type counterparts. Such results suggest that PnFL-2 could be involved in mediating floral induction [44]. Heterologous overexpression of Saccharum officinarum ScJAZ2 causes early flowering in transgenic Arabidopsis [45]. The flowering process and floral organ size are co-determined by cell proliferation and cell expansion. Guan et al. [46] investigated the expression of 13 TIFY genes in Phalaenopsis aphrodite and found that eight genes showed relatively high expression during the bud stage, with expression gradually decreasing after flower opening. These genes may promote flower opening by regulating cell growth within the buds. In this study, six VbTIFY genes (VbTIFY1/3/5/9/12/16) were selected, and their expression in three tissues of V. bonariensis was validated using qRT-PCR. The results demonstrated that two genes, VbTIFY12 and VbTIFY16, exhibited the highest expression levels in floral tissues, with the expression abundance of VbTIFY16 being 12.7-fold higher than that of VbTIFY12. Phenotypic analysis revealed that both petal size and leaf area of VbTIFY16-OE plants were significantly smaller than those of WT plants. Further cytological observations indicated that the petal cell diameter and cell area in VbTIFY16-OE lines were lower than those in WT plants. Meanwhile, there was no significant difference in the total number of epidermal cells at the petal base, which suggested that the inhibition of petal size was mainly caused by the reduction of cell expansion. Based on the above findings, we speculate that VbTIFY16 may participate in the floral development of V. bonariensis by regulating cell expansion and growth in floral tissues.

Although this study provides a preliminary bioinformatic analysis of the TIFY gene family in a single genome of V. bonariensis, certain limitations remain: Firstly, given that V. bonariensis is an autotetraploid species (4n), this study focused exclusively on the analysis of TIFY genes within a single subgenome (n) and did not include members from the remaining three subgenomes. Consequently, these findings only reflect the characteristics of this gene family within a partial genomic context, and cannot fully elucidate its overall evolutionary patterns in the tetraploid background, resulting in an incomplete characterization of the complete gene repertoire of the TIFY family. Future studies must integrate multi-subgenome data to fully characterize the TIFY family and understand its evolutionary dynamics in this polyploid species. Until such data are available, the conclusions drawn here, particularly regarding gene counts, phylogenetic relationships, and functional predictions, should be interpreted with this genomic context in mind.

Secondly, the depth of functional mechanism analysis remains limited. Although transcriptome profiling, qRT-PCR, and tobacco overexpression experiments confirmed that VbTIFY16 is most highly expressed in flowers and may lead to reduced petal size. While our overexpression data support a negative regulatory role for VbTIFY16 in petal size, loss-of-function validation is required to confirm this conclusion. We propose that VIGS (Virus-Induced Gene Silencing)-mediated transient knockdown of VbTIFY16 in V. bonariensis petals represents a feasible next step, as it would enable functional validation in the native species without the need for stable transformation. Such an experiment would predict that VbTIFY16-silenced flowers exhibit enlarged petals with increased cell area, directly complementing our overexpression findings. Furthermore, future studies will integrate multi-subgenome data to improve the systematic characterization of the TIFY gene family, and combine protein interaction assays with stable loss-of-function analyses via CRISPR/Cas9 and homologous transgenic systems, aiming to thoroughly reveal the molecular regulatory pathway of VbTIFY16 during petal development. As a result, the current understanding of its molecular regulatory network is still limited to phenotypic correlations, without thorough dissection at the pathway level. Integrating multi-subgenome data in future investigations will enable a more complete characterization of the entire TIFY gene family.

Moreover, mechanistic understanding can be further enhanced through systematic functional experiments, including protein-protein interaction assays to establish regulatory networks and gene editing to verify physiological functions in the endogenous V. bonariensis background (rather than in heterologous hosts such as tobacco). These methodological approaches will not only strengthen the reliability of our experimental conclusions but also establish a more holistic analytical framework for deciphering the molecular mechanisms by which the TIFY family regulates floral growth and development in polyploid plants.

5 Conclusion

This study provides a comprehensive and integrative analysis of the VbTIFY gene family in V. bonariensis, uncovering 16 family members. The investigation encompasses gene structural organization, conserve motif/domain analysis, phylogenetic inference, chromosomal localization, and cis-element profiling. Results highlighted the structural conservation of the VbTIFY family, which is associated with diverse growth/developmental processes. Expression pattern assays across tissues and cold stress treatments indicated that VbTIFY genes are involved in developmental modulation and low-temperature stress tolerance. Notably, six genes (VbTIFY01, VbTIFY03, VbTIFY05, VbTIFY09, VbTIFY12, and VbTIFY16) exerts a positive regulatory effect on different tissues during plant growth and development. Functional validation via VbTIFY16-OE in N. benthamiana showed negative regulation of petal and leaf size. It is expected that the results of this study can be to further clarify the functional characteristics of TIFY family genes in V. bonariensis and to use TIFY genes to improve the quality of V. bonariensis overall.