1. Introduction
Flower structures represent the most complex and advanced structures in the plant kingdom. The typical five whorls of a bisexual flower in angiosperms include the sepals, petals, stamens, carpels, and ovules [1]. However, there is considerable variation in the size, color, morphology, and arrangement of these floral organs among different plant groups [2]. The significant morphological and structural differences in flowers across species have attracted the great interest of researchers.
Flower development in angiosperms includes three stages: floral induction, flower initiation, and organ development [3,4]. These stages were controlled by numerous genes, including homeotic genes expressed at specific times and locations [5]. In the 1990s, Coen and Meyerowitz proposed the ‘ABC model’ of flower organ development [6,7], which was subsequently refined to the widely accepted ‘ABCDE model’ [8,9,10]. In the ‘ABCDE model’, the genes controlling the formation of the five floral whorls were categorized into five classes: A, B, C/D, and E, which interact each other to regulate the floral structures [5]. These A, B, C/D, and E genes encode transcription factors that form protein complexes, bind to deoxyribo nucleic acid (DNA), and modulate the expression of genes controlling floral organ characteristics [5]. According to this model, sepal identity was determined by A and E class protein complexes; petal formation was regulated by A, B, and E class proteins; stamen identity was determined by B, C/D, and E class protein; carpel formation was regulated by C/D and E class proteins [11,12]. In Arabidopsis thaliana, Class A genes include APETALA1 (AP1) and APETALA2 (AP2); Class B genes include APETALA3 (AP3) and PISTILLATA (PI); Class C genes include AGAMOUS (AG), SPATULA (SPT), and CRABS CLAW (CRC); Class D genes include SEEDSTICK (STK), SHATTERPROOF1 (SHP1), and SHATTERPROOF2 (SHP2); Class E genes include SEPALLATA1 (SEP1), SEP2, SEP3, and SEP4 [13,14]. Except for AP2, all these genes contain a highly conserved MADS domain and belong to the MADS-box gene family [8,15]. The MADS-box transcription factors play a pivotal role in the regulation of floral organ development and formation in plants.
Many studies have demonstrated that AG proteins play a crucial role in the development of reproductive organs and in governing the maintenance and termination of floral meristems [16,17]. At the sixth stage of floral organ development, the AG gene terminates the maintenance program of inflorescence meristem cells by inhibiting the expression of the key meristem maintenance gene WUSCHEL (WUS) [18]. In ag mutant plants, the upregulated expression of the WUS gene can induce uncertainty in the development process of the floral meristem [6,19]. This resulted in floral organs that contain only sepals and petals, along with an indeterminate number of whorls, and produce a “flower-in-flower” phenotype [19]. In Arabidopsis, mutations in the class C gene AtAG resulted in the transformation of stamens into petal-like structures [20,21]. In Cyclamen, the suppression of CpAG1 expression led to the conversion of stamens into petals, whereas the suppression of CpAG2 expression resulted only in the incomplete formation of stamens and carpels [22]. Despite the similarity in their protein sequences, CpAG1 and CpAG2 had distinct roles in the third and fourth floral whorls [22]. In Phalaenopsis orchids, the two AG genes, PhalAG1 and PhalAG2, belonged to the C and D lineages, respectively. PhalAG1 and PhalAG2 were expressed at significantly higher levels in floral buds and exhibited redundant functions during flower development [23]. The dynamic balance of auxin also plays a crucial role in the termination of floral meristem and the formation of the pistil [24]. The AG gene indirectly inhibits the expression of the TORNADO2 (TRN2) gene by activating the expression of CRABS CLAW (CRC), leading to the production of appropriate auxin maxima during pistil formation, thereby regulating the termination of flower meristem [25]. In addition, AG gene also ensures the expression of the AG gene in stamen or pistil by antagonizing with AP2, which promotes the normal development of reproductive organs [26,27]. The AG gene plays an important role in regulating the normal development of plant reproductive organs, and the loss of its expression may be the key to the formation of double flowers.
Clematis L., a perennial climbing plant within the Ranunculaceae family, has the reputation of being the ‘Queen of the vine’ and is one of the most popular climbing ornamental plants in the world [28,29]. The flowers of Clematis are characterized by the absence of true petals; instead, the structures resembling petals are developed from sepals, which are morphologically termed tepals [28]. Clematis flower types are categorized into three distinct forms based on the number of calyx petals and the extent of petaloid transformation of the pistil and stamen: single, semi-double, and double tepal [26]. The degree of double-tepal formation significantly impacts the ornamental appeal of these plants, and the development of double-tepal flowers has become a focal point in ornamental plant breeding due to their increasing application value.
In this study, we identified nine ABCE model genes from the transcriptome data of Clematis cv. ‘Amethyst Beauty’. We conducted a comprehensive analysis of their phylogenetic relationships, conserved protein motif structures, and gene expression profiles. Additionally, the function of the ClAG2 gene in regulating flower pattern development was verified by heterologous transformation in tobacco. The findings of this study provided a theoretical basis for elucidating the molecular mechanism underlying Clematis development regulation.
2. Materials and Methods
2.1. Plant Material
The Clematis single-tepal cultivar ‘Amethyst Beauty’ and the double-tepal cultivar ‘Alba Plena’ were planted in the greenhouse of the Genetics and Breeding Laboratory of Horticultural Plants, College of Horticulture, Fujian Agriculture and Forestry University (Fuzhou, China). Both Clematis cultivars were grown in pots using the same cultivation and management methods. The breathable and water-permeable substrate were used to ensure adequate light and avoid summer exposure. The seeds of the K326 wild-type tobacco (Nicotiana tabacum) were stored in the laboratory. Wild-type tobacco seeds were sterilized with 20% NaClO solution for 10 min under aseptic conditions. Following sterilization, the seeds were washed three times with distilled water. Then, the seeds were placed on filter paper to dry and sown on Murashige and Skoog solid medium to germinate for one month [30].
2.2. ABCE Model Genes Identification
According to the published ABCE model genes sequence of Arabidopsis, ABCE model genes related to the development of plant floral organs were screened from the transcriptome sequencing data of ‘Amethyst Beauty’. The gene structures were analyzed by Batch CD-Search (
2.3. Phylogenetic Tree Analysis
The published ABCE model homologous protein sequences of seven species were retrieved from NCBI website (
2.4. Quantitative Real-Time PCR Analysis
Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed using the ChamQ SYBR qPCR Master Mix kit (Vazyme, Nanjing, China) on the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each reaction utilized 2 μL of diluted complementary deoxyribo nucleic acid (cDNA), and the remaining reaction components and conditions were carried out according to the manufacturer’s instructions. Specific primers were designed for qRT-PCR (Table S1). ClAtpb was used as the internal reference gene. Three replicates were evaluated for each treatment. The relative expression levels were calculated using the 2−ΔΔCt method [33].
2.5. Cloning and Tobacco Transformation of ClAG2
The ribonucleic acid (RNA) was extracted by a plant total RNA extraction kit (FastPure Universal Plant Total RNA Isolation Kit) (Vazyme, Nanjing, China). Subsequently, cDNA was reverse-transcribed by HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The full coding domain sequence (CDS) of ClAG2 was cloned using the cDNA of ‘Amethyst Beauty’ as a template. The plant expression vector was constructed using an In-Fusion HD Cloning Kit (Takara, Beijing, China). Then, 35S::ClAG2 was constructed by inserting the CDS sequence of ClAG2 into the pSAK277 vector through the EcoR Ⅰ and Hind Ⅲ restriction sites. Stable transformation of the tobacco was carried out by Agrobacterium tumefaciens infection and the leaf disc method [30]. Positive seedlings were identified by polymerase chain reaction (PCR) (Figure S2). Sepals, petals, pistils, and stamens of transgenic tobacco flowers were collected separately. The expression levels of ClAG2 in various flower organs were detected using qRT-PCR.
2.6. Yeast Two-Hybrid Assay
In order to further explore the function of ClAG2, the CDS sequence of ClAG2 was inserted into the pGBKT7 vector through the EcoR Ⅰ and BamH Ⅰ restriction sites to construct pGBKT7-ClAG2. CDS sequences of ClAP3, ClPI, ClSEP3, and ClSEP4 were inserted into the pGADT7 vector to construct pGADT7-ClAP3, pGADT7-ClPI, pGADT7-ClSEP3, and pGADT7-ClSEP4 through the EcoR Ⅰ and BamH Ⅰ restriction sites. Then, the toxicity and self-activation of pGBKT7-ClAG2 were detected. The yeast two-hybrid assays of pGBKT7-ClAG2 with pGADT7-ClAP3, pGADT7-ClPI, pGADT7-ClSEP3, and pGADT7-ClSEP4 were carried out.
2.7. Statistical Analyses
Significant intergroup differences were assessed utilizing one-way analysis of variance (ANOVA) [30]. Post hoc pairwise comparisons were conducted employing Tukey’s test, with significance set at p < 0.05 [30]. Two-group comparisons were executed utilizing a two-tailed Student’s t-test. The statistical analyses were carried out using SPSS version 28.0.1.1 software.
3. Results
3.1. Morphological Characteristics of Single- and Double-Tepal Flowers of Clematis
The floral structure of Clematis comprises sepals, stamens, pistils, and ovules, with the absence of true tepals. ‘Alba Plena’ is a natural double-tepal variation of Clematis, belonging to the Florida line (F-line). ‘Amethyst Beauty’ is a single-tepal cultivar, also belonging to the F-line (Figure 1A). Observations revealed that both ’Amethyst Beauty’ and ’Alba Plena’ possess six sepals. While ’Amethyst Beauty’ exhibits typical stamens and pistils, ’Alba Plena’ displays stamens and pistils that are transformed into petaloid structures (Figure 1B).
3.2. Identification of ABCE Model Genes in Clematis
We identified nine ABCE model genes from the transcriptome of ‘Amethyst Beauty’ (Table 1) and analyzed their protein sequences for conserved domains and motifs (Figure 2A). Among these, Gene.89822 (ClAP2) was a member of the AP2/ERF family, while the remaining genes belong to the MADS-box family. A phylogenetic tree was constructed by clustering these nine proteins with ABCE model proteins from other species (Figure 2B). Gene.53960 (ClAP1) grouped with PhAP1 and AtAP1, and Gene.89822 (ClAP2) grouped with AtAP2 and PhAP2, both of which are classified as A-Class. Gene.60345 (ClAP3) clustered with AtAP3 and pMADS1, and Gene.56658 (ClPI) clustered with AtPI and PhGOL1, which are B-Class genes. Gene.101921 (ClAG1) and Gene.78312 (ClAG2) were clustered with AtAG and pMADS3, which are C-Class genes. Gene.68269 (ClSEP1), Gene.55466 (ClSEP3), and Gene.63127 (ClSEP4) were clustered with FBP2, AtSEP3, and AtSEP4, belonging to E-Class genes. D-Class genes were not distinctly identified, possibly because they belong to the evolutionary branch of C-Class genes, and there is no clear distinction between D-class genes and C-class genes in Clematis.
3.3. Expression Level Analysis of ABCE Model Genes in Single- and Double-Tepal Flowers of Clematis
The expression levels of ABCE model genes were detected in the flower organs of ‘Amethyst Beauty’ and ‘Alba Plena’, separately (Figure 3A). The results showed that the expression levels of ClPI, ClSEP1, and ClSEP3 were higher in the sepals of ‘Amethyst Beauty’ and ‘Alba Plena’. ClAP3, ClPI, ClAG2, ClSEP1, and ClSEP3 were expressed in stamens, and ClAG1 was expressed in pistils of ‘Amethyst Beauty’. The expression levels of ClPI, ClSEP1, and ClSEP3 were higher in the ‘Alba Plena’ petalody part. Considering that the formation of double-tepal flowers of Clematis is related to stamen and pistil petalization, the expression levels of ABCE model genes in stamens, pistil, and petalody parts were compared (Figure 3B). It was found that the expression levels of ClAP1, ClAP2, ClPI, ClAG1, and ClSEP1 in petaloid parts were higher than those in stamens, while the expression levels of ClAP3 and ClAG2 in petaloid parts were lower than those in stamens. There was no difference between the expression levels of ClSEP3 and ClSEP4 in stamens and petaloid parts. Combined with tissue specific analysis of the above gene expression. We believe that ClAG1 is related to the normal development of the pistil, ClAP3 and ClAG2 may specifically regulate the stamen development of clematis, and they may be related to the formation of double-tepal flowers of Clematis.
3.4. Overexpression Analysis of ClAG2 in Tobacco
To investigate the function of ClAG2, we cloned the ORF sequence of ClAG2 from the cDNA of ’Amethyst Beauty’ (Figure S1). The ORF sequence of ClAG2 is 681 bp, encoding 227 amino acids.
ClAG2 was further transferred to tobacco by the Agrobacterium-mediated method. We found significant differences in flower morphology between transgenic tobacco and wild-type tobacco (WT) (Figure 4A,B). The corolla of WT was funnel-shaped, measuring 22–23 mm in diameter and 51–52 mm in tube length, with relatively elongated, pink floral lobes. In contrast, the 35S::ClAG2 transgenic tobacco flowers have a thinner and shorter corolla tube, with a diameter of 10–12 mm and a length ranging from 27 mm to 49 mm. The floral lobes of these transgenic flowers converged inward and were light pink in color. In the genital organs, the length of style and capillament of transgenic tobacco was significantly shorter than that of WT. In addition, the anthers of transgenic tobacco showed wilting and little or no pollen. qRT-PCR analysis showed that the expression of ClAG2 in each flower organ of transgenic tobacco was significantly higher than that of the WT (Figure 4C). This means that the abnormal phenomena in transgenic tobacco flowers may be caused by high expression of ClAG2.
3.5. Interaction Analysis Between ClAG2 and ClAP3, ClPI, ClSEP3 and ClSEP4
According to the tetramer model, Class B, C, and E proteins together regulate the development of stamens, and Class C and E proteins regulate the development of the pistil. Therefore, the interaction ability of ClAG2 with ClAP3, ClPI, ClSEP3, and ClSEP4 were verified. Toxicity and self-activation tests showed that pGBKT7-ClAG2 was non-toxic to yeast and had no self-activation (Figure S3). The results of yeast two-hybrid showed that ClAG2 can interact with E-Class proteins ClSEP3 and ClSEP4 (Figure 5).
4. Discussion
For ornamental plants, flower morphology is one of the most important phenotypes that determine the economic value of ornamental plants [34]. Therefore, the molecular regulation mechanism of flower development has always been the focus of research. For decades, the ABCE model has served as a framework for understanding flower development [35,36]. Most studies on flower development have focused on ABCE model genes. Clematis is an important ornamental plant known for its rich variety of flower colors and shapes, earning it the title ‘Queen of Vines’ [29]. However, the key mechanisms regulating the formation of rich flower patterns of Clematis are still unclear. Therefore, to identify the members of the Clematis ABCE model gene and study their expression profile and function, it is of great significance to understand the regulation mechanism of the Clematis rich flower pattern.
The bisexual flowers of angiosperms have a five-whorled structure, including sepals, petals, stamens, carpels, and ovule, which are formed by the synergistic action of class A, B, C, and E genes [1,37,38]. During the differentiation of floral organs, the A class gene AP1 determines the development of the first and second whorls (sepals and petals) [39,40]. Loss of AP1 function leads to failure in the initiation of floral organ differentiation or to homeotic transformations, primarily resulting in the development of sepal-like organs instead of sepals and a reduction in petal number [41]. Mutations in B class genes can cause alterations in the second and third whorls (petals and stamens), leading to petals transforming into sepals and stamens into carpels [42]. The genes of the C class are essential for the normal development of stamens and carpels, serving as core regulators of their formation [43,44,45]. The genes of the E class maintain the proper development of the five whorls of floral structures by interacting with the other four classes of genes [46,47]. In this study, a total of nine ABCE model genes were identified, including two A-class genes, two B-class genes, two C-class genes, and four E-class genes. Through qRT-PCR experiments and comparison of expression levels in single-tepal cv. ‘Amethyst Beauty’ and double-tepal cv. ‘Alba Plena’, it was found that ClPI, ClSEP1, and ClSEP3 may be key regulatory genes for sepal development in Clematis. ClAP3, ClPI, ClAG2, ClSEP1, and ClSEP3 were potential genes involved in stamen development, and ClAG1 plays an indispensable role in pistil formation. The high expression level of ClPI, ClSEP1, and ClSEP3 in the petalody parts of ‘Alba Plena’ may be crucial for the development of double-tepal flower Clematis.
Clematis can be divided into three types based on the degree of petalization of their stamens and carpels: single, semi-double, and double-flower [48]. Double-tepal flowers exhibit significant petalization of both stamens and carpels. Studies have shown that class C genes play an important role in regulating the formation of stamen and carpels; the loss of their functions will promote the transition of stamen and carpel toward petals, thus promoting the formation of double flowers [43,44,45]. In Arabidopsis, the AG gene played a crucial role in the development of floral meristems and the formation of reproductive organs [49]. The Arabidopsis ag mutants exhibit a transformation of stamens into petals. In addition, AG and aptala2 (AP2) specify the formation of reproductive organs (stamen and pistil) and perianth (sepals and petals) in an antagonistic manner [26,27]. In Cyclamen persicum, CpAG1 and CpAG2 were expressed in stamen and carpel. CpAG1 is mainly involved in controlling stamen development, while CpAG2 is mainly involved in specifying carpel identity and terminating meristem activity [22]. In Phalaenopsis orchids, two AG genes, PhalAG1 and PhalAG2, belong to the C and D lineages, respectively. The expression levels of PhalAG1 and PhalAG2 in flower buds were much higher than those in vegetative organs and played a redundant role in flower development [23]. Similarly, we identified two ClAG genes in Clematis. Among them, ClAG1 is specifically expressed in the pistil, while ClAG2 is expressed in both stamens and pistil. This suggested that ClAG1 and ClAG2 jointly regulate the pistil development of Clematis, while stamens were regulated by ClAG2.
Delaying or inhibiting the development of petals and sepals is another conserved function of the AG gene, which is usually caused by antagonism between the AG gene and AP2 [50]. In Arabidopsis thaliana, AP2 represses the expression of AG in the first and second whorls of floral organs, while AG inhibits the expression of AP2 in the third and fourth whorls [51]. Similar reports have been documented in many plants. Overexpression of the DcaAG gene from carnations in Arabidopsis thaliana resulted in abnormal development of the third and fourth whorls in transgenic plants, with inhibited petal growth [52]. The heterologous overexpression of TeAG from marigolds in Arabidopsis resulted in curled rosette leaves and early flowering, with a disruption in the formation of normal sepals and petals, significantly suppressing petal development [53]. In Ilex verticillata, IlveAG is highly expressed in the stamens, carpels, and sepals of both male and female flowers [54]. IlveAG can functionally replace endogenous AG to rescue the stamen and carpel development in the Arabidopsis thaliana ag-1 mutant. Additionally, the expression of IlveAG can inhibit the development of sepals and petals in both wild-type and ag-1 Arabidopsis thaliana [54]. Our study showed a similar phenomenon. Overexpression of ClAG2 inhibited the development of the corolla tube of tobacco flowers. This indicated that the ClAG2 gene has similar functions to class C genes with the other species.
As a transcription factor, AG has been reported to promote stamen and pistil development in flower organs by interacting with other ABCE model genes. AG proteins can interact directly with E-class proteins, whereas other transcription factors cannot interact with AG. It is necessary to form a complex with AG protein through E-class protein SEP3 as a bridge [55]. The B-class proteins AP3 and PI cannot directly interact with AG [56]. They rely on the E protein SEP3 to interact with AG [57]. Therefore, the dimer formed between AG and SEP3 proteins is crucial for floral morphogenesis. Yeast two-hybridization experiments showed that ClAG2 could interact with ClSEP3 and ClSEP4 but not directly with ClAP3 and ClPI, which is consistent with the findings described above. In addition, the low expression of the ClAG2 gene in the double-tepal cv. ‘Alba Plena’ may be a primary factor leading to the transformation of stamens and pistils into petalody structures. However, the function of ClAG2 in the formation of double flowers in Clematis requires further validation.
5. Conclusions
In this study, a total of nine ABCE genes were identified from the transcriptome of Clematis. The results of qRT-PCR and tobacco transgenic assays showed that ClAG2 is the key gene in regulating the normal development of stamen and carpels of Clematis. The low expression of ClAG2 in double-tepal Clematis may be the key factor promoting the formation of double-tepal Clematis flowers. Our results have deepened people’s understanding of the mechanism of Clematis flower development and laid a theoretical foundation for further analysis of the molecular mechanism of Clematis double flower formation.
X.C., P.Z. and L.Z.: conceptualization and writing. N.G., Y.Z. and X.H.: validation. P.Z. and X.C.: methodology. X.C.: formal analysis. L.Z.: editing and reviewing. All authors have read and agreed to the published version of the manuscript.
The transcriptome data used in this article are available for download from NCBI (
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 1. Flower morphology of Clematis. (A) Flower morphology of ‘Amethyst Beauty’ and ‘Alba Plena’; (B) Observation of longitudinal sections and floral organs of ‘Amethyst Beauty’ and ‘Alba Plena’. Scale bar = 10 mm.
Enlarge this image.Figure 2. Structure analysis and cluster analysis of ABCE model protein in Clematis. (A) Protein structure of ABCE model genes in Clematis; (B) Phylogenetic tree of ABCE model genes of Clematis and other species, ABCE model genes from Clematis are marked in red.
![View Image - Figure 3. Expression level analysis of ABCE model genes in Clematis. (A) Expression levels of ABCE genes in flower organs of ‘Amethyst Beauty’ and ‘Alba Plena’; Data are mean ± SD (n = 3). Different letters denote significant differences (p [less than] 0.05, one-way ANOVA). (B) Comparison of expression levels of ABCE model genes in stamens and pistils of ‘Amethyst Beauty’ and petalody parts of ‘Alba Plena’. Data are mean ± SD (n = 3). The ns indicates no significance statistical significance at p [greater than] 0.05, * indicates statistical significance at p [less than] 0.05, ** indicates statistical significance at p [less than] 0.01, *** indicates statistical significance at p [less than] 0.0001, and **** indicates statistical significance at p [less than] 0.00001 (t-test). Note: ‘Z’ means ‘Amethyst Beauty’ and ‘L’ means ‘Alba Plena’.](https://media.proquest.com/media/hms/THUM/6/o77Mb?_a=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%3D%3D&_s=dFy3s%2BIhmgHRZGutTIQxC%2BEZ5OQ%3D "View Image - Figure 3. Expression level analysis of ABCE model genes in Clematis. (A) Expression levels of ABCE genes in flower organs of ‘Amethyst Beauty’ and ‘Alba Plena’; Data are mean ± SD (n = 3). Different letters denote significant differences (p [less than] 0.05, one-way ANOVA). (B) Comparison of expression levels of ABCE model genes in stamens and pistils of ‘Amethyst Beauty’ and petalody parts of ‘Alba Plena’. Data are mean ± SD (n = 3). The ns indicates no significance statistical significance at p [greater than] 0.05, * indicates statistical significance at p [less than] 0.05, ** indicates statistical significance at p [less than] 0.01, *** indicates statistical significance at p [less than] 0.0001, and **** indicates statistical significance at p [less than] 0.00001 (t-test). Note: ‘Z’ means ‘Amethyst Beauty’ and ‘L’ means ‘Alba Plena’.")
Enlarge this image.Figure 3. Expression level analysis of ABCE model genes in Clematis. (A) Expression levels of ABCE genes in flower organs of ‘Amethyst Beauty’ and ‘Alba Plena’; Data are mean ± SD (n = 3). Different letters denote significant differences (p [less than] 0.05, one-way ANOVA). (B) Comparison of expression levels of ABCE model genes in stamens and pistils of ‘Amethyst Beauty’ and petalody parts of ‘Alba Plena’. Data are mean ± SD (n = 3). The ns indicates no significance statistical significance at p [greater than] 0.05, * indicates statistical significance at p [less than] 0.05, ** indicates statistical significance at p [less than] 0.01, *** indicates statistical significance at p [less than] 0.0001, and **** indicates statistical significance at p [less than] 0.00001 (t-test). Note: ‘Z’ means ‘Amethyst Beauty’ and ‘L’ means ‘Alba Plena’.
![View Image - Figure 4. Overexpression analysis of ClAG2 in tobacco. (A) Comparison of flower morphology between transgenic tobacco and wild-type tobacco (WT). Scale bar = 10 mm. (B) Statistics of floral organ size of transgenic tobacco and transgenic tobacco. (C) The expression level of ClAG2 in different floral organs of transgenic tobacco. Data are mean ± SD (n = 3). The ** indicates statistical significance at p [less than] 0.01, *** indicates statistical significance at p [less than] 0.001, and **** indicates statistical significance at p [less than] 0.0001 (t-test).](https://media.proquest.com/media/hms/THUM/8/o77Mb?_a=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%3D%3D&_s=4iTVqDdNnR4K8o3Bv1FQTved2Rk%3D "View Image - Figure 4. Overexpression analysis of ClAG2 in tobacco. (A) Comparison of flower morphology between transgenic tobacco and wild-type tobacco (WT). Scale bar = 10 mm. (B) Statistics of floral organ size of transgenic tobacco and transgenic tobacco. (C) The expression level of ClAG2 in different floral organs of transgenic tobacco. Data are mean ± SD (n = 3). The ** indicates statistical significance at p [less than] 0.01, *** indicates statistical significance at p [less than] 0.001, and **** indicates statistical significance at p [less than] 0.0001 (t-test).")
Enlarge this image.Figure 4. Overexpression analysis of ClAG2 in tobacco. (A) Comparison of flower morphology between transgenic tobacco and wild-type tobacco (WT). Scale bar = 10 mm. (B) Statistics of floral organ size of transgenic tobacco and transgenic tobacco. (C) The expression level of ClAG2 in different floral organs of transgenic tobacco. Data are mean ± SD (n = 3). The ** indicates statistical significance at p [less than] 0.01, *** indicates statistical significance at p [less than] 0.001, and **** indicates statistical significance at p [less than] 0.0001 (t-test).
Enlarge this image.Figure 5. Yeast two-hybrid of ClAG2 with ClAP3, ClPI, ClSEP3, and ClSEP4. DDO: SD-Leu-Trp medium, TDO: SD-Leu-Trp-His medium, QDO: SD-Leu-Trp-His-Ade medium, QDO+X-α-Gal: SD-Leu-Trp-His-Ade medium + X-α-Gal. The ‘pGBKT7-53 + pGADT7-T’ were expressed in yeast as a positive control; the ‘pGBKT7 + pGADT7’ and ‘pGBKT7-ClAG2 + pGADT7’ were expressed in yeast as negative controls.
Basic information of the ABCE model gene in Clematis.
| Gene Name | Gene Identity Document | Amino Acid | Theoretical Isoelectric Point | Molecular Weight | Predicted Location(s) |
|---|---|---|---|---|---|
| ClAP1 | Gene.53960 | 237 aa | 7.85 | 26,514.29 | Nucleus. |
| ClAP2 | Gene.89822 | 509 aa | 7.14 | 56,066.67 | Cytoplasm. Nucleus. |
| ClAP3 | Gene.60345 | 222 aa | 8.45 | 26,558.78 | Nucleus. |
| ClPI | Gene.56658 | 209 aa | 9.11 | 24,327.89 | Nucleus. |
| ClAG1 | Gene.101921 | 226 aa | 9.14 | 25,977.77 | Nucleus. |
| ClAG2 | Gene.78312 | 226 aa | 9.32 | 25,780.05 | Nucleus. |
| ClSEP1 | Gene.68269 | 249 aa | 9.28 | 28,547.27 | Nucleus. |
| ClSEP3 | Gene.55466 | 239 aa | 9.03 | 27,405.96 | Nucleus. |
| ClSEP4 | Gene.63127 | 238 aa | 5.77 | 23,151.05 | Nucleus. |
Note: aa represents amino acid.
Supplementary Materials
The following supporting information can be downloaded at
References
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