The Functional Verification of CmSMXL6 from Chrysanthemum in the Regulation of Branching in Arabidopsis thaliana

Abstract

The development of branching plays a pivotal role in the cultivation of ornamental chrysanthemums, as it dictates the ultimate morphology and quality of the plants. Strigolactones (SLs) are associated with apical dominance to indirectly inhibit shoot branching. Chrysanthemum morifolium ‘Baltasar’ in this study was subjected to treatment with three hormones: auxin (IAA), 6-BA, and GR24. Following the exogenous application of GR24 and IAA, a significant reduction in both the length and quantity of lateral buds on chrysanthemums was observed. Additionally, there was a notable down-regulation in the expression levels of CmPIN1 (associated with auxin transport) and CmIPT3, which is involved in cytokinin (CK) synthesis. After the application of 6-BA, there was a significant increase in both the length and quantity of lateral buds on chrysanthemums. Subsequently, the separate application of IAA and 6-BA to C. morifolium ‘Baltasar’ notably induced the expression of CmMAX1, a gene involved in the biosynthesis of strigolactones, and CmSMXL6, a gene associated with the signaling pathway of SLs, suggesting a negative regulatory role for SLs and auxin in chrysanthemum lateral buds, while CK demonstrated positive regulation. Cloning and expression analysis of CmSMXL6, a member of the D53/SMXL gene family in chrysanthemum, revealed its up-regulation following GR24 treatment, peaking at 9 h. The overexpression of CmSMXL6 in Arabidopsis thaliana promoted increased numbers of primary and secondary branches. In transgenic lines, genes associated with SLs synthesis (AtMAX1, AtMAX2, and AtMAX3) exhibited varying degrees of down-regulation, while the branching-inhibitory gene AtBRC1 also displayed decreased expression levels. These findings suggest that CmSMXL6 plays a role in promoting branching.

1. Introduction

Plant architecture is an important agronomic trait, comprising plant height, branching, leaf shape, and inflorescence type. Branching, as an important component of the aboveground plant architecture, plays a crucial role in crop production, plant morphogenesis, and landscaping with ornamental plants. The regulation of plant branching is influenced by a complex interplay of internal and external factors. To date, a significant amount of research has been focused on the impact of endogenous factors (such as hormones and genetic factors) on plant branching [1,2]. Plant hormones are a class of trace signal molecules synthesized within the plant, which can regulate the growth and development of plant axillary buds at low concentrations [3]. In particular, plant hormones such as auxin (IAA), cytokinin (CK), gibberellins (GAs), brassinolide (BR), and strigolactones (SLs) have a significant impact on plant branching [4]. SLs are a class of sesquiterpene compounds that can induce the germination of Striga asiatica seeds and promote the branching of arbuscular mycorrhiza fungi (AMF). Subsequent research has indicated that SLs play important regulatory roles in multiple growth and development processes in plants, with the most significant function being the direct or indirect suppression of lateral bud growth [5]. The negative regulatory role of SLs in plant branching has been observed in several species, including Pisum sativum L. (Fabaceae) [6], Oryza sativa L. (Poaceae) [2], and Malus pumila Mill. (Rosaceae) [7]. GR24 is a synthetic analog of strigolactone. Studies have shown a reduction in the number and biomass of lateral branches in Quercus mongolica Fisch. (Fagaceae) seedlings when treated with different concentrations of GR24 under all experimental conditions [8]. Following the application of 1 μM GR24 to O. sativa, the complete inhibition of tillering was observed [9]. As a series of SLs-insensitive mutants have been identified across various species, key components of the SLs signal transduction pathway have been successively cloned.

The majority of genes related to the synthesis and signal transduction of strigolactones were identified through studies on mutant variants with branching abnormalities [10]. Through the study of branching mutants, genes related to MAX3/MAX4/MAX1/MAX2/AtD14 in Arabidopsis thaliana, D27/D17/HTL17 in O. sativa, and DAD1/2/3/PhMAX2A in Petunia ×hybrida (Hook.) Vilm. (Solanaceae) have been identified [11]. The mutants max3 and htd1 exhibit an enhanced branching phenotype [12]. The D53/SMXL protein family is a group of proteins involved in the signal transduction process of SLs. Mutants with the functional loss of D53-homologous genes in A. thaliana, including SMAX1-LIKE6 (SMXL6), SMXL7, and SMXL8, all exhibit the suppression of the max2 phenotype in the SLs pathway [13]. This suggests that D53/SMXL proteins act as inhibitors to regulate leaf morphology and plant branching, indicating their role in negatively regulating SLs signal reception and response processes [14]. As research has advanced, homologous genes of the D53 gene family have been identified in a variety of plants, including A. thaliana, O. sativa, Gossypium spp., Populus spp., and P. sativum [15]. The PagD53 gene in P. alba exhibits high expression levels in axillary buds and related nodes [16]. The PagD53 protein regulates plant branching development through signal transduction mediated by SLs [17]. In P. sativum, the D53/SMXL gene family comprises three members, namely, PsSMXL6, PsSMXL7, and PsSMXL8. Among these, the PsSMXL7 protein in peas has been found to promote bud growth through the inhibition of PsBRC1 expression [18]. And, D53 in O. sativa can interact with OsBZR1 to regulate branching [19]. In addition to their impact on branching, D53/SMXLs play a negative regulatory role in drought stress. AtSMXL6/7/8 were involved in Arabidopsis’s response to drought, with the triple mutant Atsmxl6/smxl7/smxl8 demonstrating greater drought tolerance than the wild type [20].

D53/SMXLs act as inhibitors of SLs signal transduction and can also regulate other hormones through this pathway. The protein structure of D53/SMXLs contains a conserved EAR domain involved in suppressing SLs signaling. Studies have shown that the EAR motif of the AtSMXL6 protein can suppress the expression of the BRC1 gene by recruiting TPR2, thereby inhibiting abscisic acid (ABA) synthesis and promoting plant branching [21]. Numerous studies have demonstrated that SLs, auxin, and CK are involved in feedback regulation during the process of plant branching development. Mutants such as max of A. thaliana and rms of P. sativum exhibit a substantial decrease in CK content compared to wild-type plants [22]. In P. sativum, apical pruning treatment leads to reduced levels of auxin and increased levels of CK in the xylem sap, resulting in the downregulation of the transcriptional activity of the SL synthesis genes RMS1 and RMS5 [23]. Both SLs and CK can directly regulate the development of lateral buds, possibly by controlling a common node, namely, the BRC1 gene [24]. A reduction in the relative expression level of PsBRC1 was observed in an SLs mutant of P. sativum. The treatment of axillary buds of P. sativum with the synthetic analog GR24 for 6 h resulted in further suppression of PsBRC1 expression, while exogenous CK treatment down-regulated BRC1 expression and promoted the expression of genes associated with the cell cycle and PIN genes [25]. The foliar spraying of Festuca arundinacea Lilj. (Poaceae) seedlings with different concentrations of GA resulted in a mutual antagonism between GA3 and CK, leading to a deceleration in the transformation of axillary buds into tillers [26].

Chrysanthemum morifolium Ramat. ex Hemsl (Asteraceae) is one of the world’s four major cut flowers, with significant economic value. Branching in chrysanthemums is crucial for plant architecture, and the cost associated with managing and controlling branching accounts for approximately one-third of total cultivation expenses in chrysanthemum production. SLs, as negative regulators of plant lateral branching, have become a focal point in the regulation of branch development. However, the specific mechanisms underlying the interaction between SLs and other hormones remain unclear. The SMXL gene family, with dual inhibitory functions in the SLs signaling pathway, plays a crucial role in plant branching. However, most research has been concentrated on model plants, and there have been no reports on its study in chrysanthemums. Therefore, this study aimed to clone and conduct a functional expression analysis of the D53/SMXL-homologous gene of A. thaliana in C. morifolium ‘Baltasar’, as well as to investigate the biological function of the D53/SMXL-like protein family in chrysanthemum. The results are of great significance in providing a theoretical foundation for the improvement of lateral branch growth characteristics in chrysanthemum.

2. Materials and Methods

2.1. Experimental Site and Planting Material

The C. morifolium ‘Baltasar’ used in the experiment was collected from Guangzhou Houde Agricultural Technology, Guangzhou, China. Cuttings from strong-growing chrysanthemums were used for rooting. After rooting, the chrysanthemums were transplanted into pots filled with a mixture (2:1) of commercial potting soil and vermiculite, and plants that had been planted for 25 days were used as experimental materials. All the plants were cultivated in a growth chamber with a controlled environment (16 h of light/8 h of darkness).

2.2. Exogenous Hormone Treatment

The top buds of all chrysanthemums were quickly cut off from the plants with disinfected blades for topping treatment. Twenty-five-day-old chrysanthemum plants were sprayed with different hormone treatments, including 100 μM IAA, 10 μM GR24, or 100 μM 6-BA (Sigma-Aldrich, St. Louis, MO, USA). For the control treatment, the plants were sprayed with distilled water. The plants were sprayed every three days for a total of 3 times, and branching phenotypic characteristics, such as the length and diameter of lateral buds, were observed on the 10th day. Each treatment was set with 15 replicates. And, the stems were collected 10 days after different hormone treatments for RNA extraction to detect the expression of key genes involved in bud growth and hormones. In addition, the stems were collected 0, 1, 3, 6, 9, and 12 h after GR24 treatment, and different tissue samples (stem, root, leaf, node, and terminal bud) were harvested to explore the expression of CmSMXL6 in C. morifolium ‘Baltasar’.

2.3. Morphological Measurements

For phenotypic investigation, the length and growth rate of lateral buds were measured. The methods of measuring the length of lateral buds referred to Martin-Trillo et al. [27]. The node where the first lateral bud emerged after apical removal was considered the initial node for counting lateral buds. Subsequently, the lengths of the lateral buds were measured at 15 nodes sequentially and labeled as the 1st- to 15th-node lateral buds. Lateral buds exceeding 1 cm in length were considered valid, while those measuring 1 cm or less were deemed non-existent. Each treatment comprised 15 biological replicates.

2.4. RNA Extraction and qPCR

To analyze the expression of genes related to bud growth, like BRC1 and DRM1, and other key genes associated with hormones, including PIN1, TIR3, IPT3, MAX1, and SMXL6, in hormone-treated lines, the stems of 25-day-old chrysanthemum subjected to different hormone treatments, including 100 μM IAA, 10 μM GR24, and 100 μM 6-BA, and the control treated with distilled water were harvested to extract RNA. In addition, the stems after treatment with GR24 for different durations (0, 1, 3, 6, 9, and 12 h) and different tissues (stem, root, leaf, node, and terminal bud) were collected to explore the expression of CmSMXL6. The total RNA was extracted using the Quick RNA isolation Kit (Huayueyang Biotechnology, Beijing, China). Then, 1 μg of total RNA was transcribed into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser Perfect Real Time (TakaRa, Kusatsu, Japan). The procedure for qPCR and the use of CmEF1α in chrysanthemum as an internal reference are consistent with Wang et al. [28]. The 2^−∆∆Ct method was used to calculate relative gene expression levels. And, three biological replicates were used per sample. Meanwhile, an analysis of the expression of genes related to SLs signaling pathways and branching growth in the CmSMXL6 transgenic line of A. thaliana was conducted, including AtMAX1, AtMAX2, AtMAX3, and AtBRC1. Each sample was represented by three biological replicates. The results were analyzed using PikoReal™ 2.0 software and Microsoft Excel 2021. All primer pairs used for qPCR are listed in Table S1.

2.5. Isolation of CmSMXL6 and Sequence Analysis

The Chrysanthemum nankingense genome database was used to obtain the sequence information of CmSMXL6 (http://210.22.121.250:8880/asteraceae/homePage (accessed on 4 July 2024)). The full-length ORF sequence of CmSMXL6 was cloned with the primer pair CmSMXL6-F and CmSMXL6-R from the cDNA of C. morifolium ‘Baltasar’. The SMXL6 protein sequences from various species were retrieved from the NCBI database and analyzed using MEGA 11 software to construct a phylogenetic tree through the neighbor-joining method (NJ) with 1000 bootstrap replicates.

2.6. Subcellular Localization

For subcellular localization experiments, the pNC-Green-SubN vector with a fusion GFP tag was used as the localization expression vector. The ORF of CmSMXL6 was amplified using the primer pNC-CmSMXL6-F/R (Table S1). The PCR product was ligated with the pNC-Green-SubN plasmid, which is a vector consistent with that used in Wang et al. [28], resulting in the generation of the pNC-Green-SubN-CmSMXL6 plasmid, which was then transformed into Escherichia coli E. (Enterobacteriaceae) DH5 α. Kanamycin was employed for positive clone selection. Following the confirmation of correct sequencing, high-concentration plasmids were extracted using Omega USA’s E.Z.N.A. Plasmid Mini kit I (D6943) and subsequently transformed into Agrobacterium tumefaciens H. (Rhizobiaceae) containing pSoup. Finally, the 35S::GFP-CmSMXL6 fusion was constructed for subcellular localization. To determine the localization of CmSMXL6, the constructed pNC-Green-SubN-CmSMXL6 plasmid was introduced into A. tumefaciens GV3101 via injection and then inoculated onto the leaves of Nicotiana tabacum L. (Solanaceae). The injected tobacco plants were cultivated under dark conditions for 16 h, followed by a transfer to standard growth conditions (16 h of light/8 h of darkness). Following transformation, cells were stained with DAPI, and the fluorescence signals were observed under a confocal laser scanning microscope, Leica TCS SP8 (Leica, Wetzlar, Germany), after being cultured for 48–72 h.

2.7. Transgenic Arabidopsis Phenotype Observation

To validate the function of CmSMXL6 in A. thaliana, the pNC-Cam1304-MCS35S vector was selected as an overexpression vector. The full-length coding sequence of CmSMXL6 was cloned into the pNC-Cam1304-MCS35S vector. The vector was introduced into the flowers of wild-type A. thaliana using the freeze–thaw transformation method mediated by A. tumefaciens GV3101. The presence of the transgenic plants was confirmed through PCR amplification using specific primers (Table S1). T2 transgenic A. thaliana seeds were sown on 1/2 MS solid medium supplemented with 50 mg/L of hygromycin, while the wild-type (Col-0) seeds were sown on 1/2 MS medium as a control without hygromycin. After 2 days at 4 °C, the donor plants were transferred to a growth chamber and cultivated under long-day conditions (16 h light/8 h dark) at a temperature of 22 ± 2 °C. Subsequently, both the transgenic and control plants were grown at 22 °C under a photoperiod of 12 h light/12 h dark. Wild-type A. thaliana and CmSMXL6-overexpressing lines were selected, and the number of rosette leaves and the primary and secondary branching numbers of A. thaliana were statistically analyzed.

2.8. Statistical Analysis

The number of branches and bud diameter were determined by statistical variance analysis using the IBM SPSS Statistics 21.0 software. The relative expression levels of key genes were analyzed using PikoReal™ 2.0 software and Microsoft Excel. Data were analyzed for significance using Duncan’s method. The differences between mean values were separated at a level of p < 0.05. Origin 17.0 was used for mapping.

3. Results

3.1. Phenotypic Analysis of Chrysanthemum Branching Following Hormone Treatment

Hormones play a crucial role in influencing the lateral branch growth of plants. To investigate the impact of auxins, cytokinins, and SLs on chrysanthemum lateral branch growth, various plant hormone treatments were administered to chrysanthemum plants. As shown in Figure 1A, the number of branches on chrysanthemum plants treated with 100 µM 6-BA was significantly higher than that on the control. In contrast, both the treatments with 100 µM IAA and 10 µM GR24 exhibited a certain degree of inhibitory effect on branching compared to the control, with GR24 demonstrating a more pronounced inhibitory effect. The analysis of the lateral bud diameter revealed that chrysanthemum buds treated with 100 µM 6-BA exhibited a significantly larger diameter compared to the control group, whereas those treated with 100 µM IAA and 10 µM GR24 showed significantly smaller diameters relative to the control (Figure 1B).

On the 10th day following treatment, an assessment of lateral bud length was performed. In the 6-BA treatment, the first-node lateral bud length of chrysanthemum exhibited a reduction compared to the control, while the second- and third-node lateral bud lengths demonstrated an increase relative to those of the control. Lateral bud lengths in both the IAA and GR24 treatments were diminished compared to those in the 6-BA treatment and the control, with a discernible suppressive impact on the second- and third-node lateral bud lengths. Specifically, within the IAA treatment, measurement of the second-node lateral bud length yielded a value of 29.31 mm, indicating a reduction of 10.5 mm when contrasted with that of the control group. Similarly, for chrysanthemums subjected to treatment with GR24, the second-node lateral bud length was measured at only 27.62 mm, 12.2 mm shorter than that of the control (Figure 1C).

3.2. Analysis of Lateral Shoot Growth Rate

Further analysis was conducted to investigate the effects of three hormones, GR24, 6-BA, and IAA, on the growth and development of lateral buds. The growth rates of the second-node lateral buds were measured at 3 d, 5 d, 8 d, and 10 d. According to Table 1, it is evident that during the initial 8 days of treatment with 6-BA, chrysanthemum lateral buds exhibited the highest growth rate, indicating a significant promotion of lateral bud development. In contrast, GR24 treatment resulted in the deceleration of the lateral bud growth rate compared to the control, with the slowest observed growth rate. The growth rate of Chrysanthemum lateral buds exhibited a significant increase at 8 d, followed by a subsequent decline at 10 d. The most pronounced reduction in the growth rate was observed following treatment with 6-BA.

3.3. Analysis of Gene Expression after Hormone Treatment

The expression patterns of genes associated with shoot branching and the key genes involved in the synthesis of the three hormones are shown in Figure 2. The genes associated with shoot branching, CmBRC1 and CmDRM1, display distinct expression patterns under various hormone treatments. The expression of the shoot branching inhibitor gene CmBRC1 was significantly up-regulated in response to both IAA and GR24 treatments, with the highest relative expression level observed following IAA treatment, which was 1.86 times that of the control group. Conversely, the down-regulation of CmBRC1 expression was evident in response to 6-BA treatment, consistent with the observed branching phenotype (Figure 2A). The expression of the CmDRM1 gene, which promotes bud differentiation, exhibited significant up-regulation in response to 6-BA treatment, whereas its expression level was markedly down-regulated following GR24 treatment (Figure 2B). The expression of the auxin transporter gene, CmPIN1, was significantly up-regulated following IAA treatment (Figure 2C). The relative expression level of the cytokinin synthesis enzyme gene CmIPT3 was significantly decreased after treatment with IAA or GR24, while it was significantly increased (by 2.08 times that in the control group) after treatment with 6-BA (Figure 2D). Furthermore, the genes involved in the biosynthesis of strigolactones, CmMAX1, and their signaling pathway, CmSMXL6, were subjected to quantitative analysis in chrysanthemum. Following treatment with three distinct hormones, both CmMAX1 and CmSMXL6 exhibited varying degrees of up-regulation in their relative expression levels (Figure 2E,F).

3.4. Isolation and Characterization of CmSMXL6

The full-length CmSMXL6 sequence was cloned using specific primers from C. morifolium ‘Baltasar’ cDNA. The sequence analysis indicates that the ORF of CmSMXL6 is 2823 bp (Figure 3A), predicting the encoding of 940 amino acid residues. The calculated molecular weight of CmSMXL6 is 104.28 kDa, with an isoelectric point of 5.76. The predicted amino acid sequence of CmSMXL6 has a high homology with the proteins of other species, which is shown in Figure 3C. A phylogenetic analysis implied that CmSMXL6 is most closely related to the proteins of Cucurbita moschata (Duchesne) Duchesne ex Poir. (Cucurbitaceae) and Juglans microcarpa Berl. (Juglandaceae) (Figure 3B).

3.5. Subcellular Localization of CmSMXL6

In order to determine the subcellular localization of the CmSMXL6 protein in living cells, the pNCGreen-CmSMXL6 vector was constructed and introduced into tobacco epidermal cells, and the GFP signal was observed. The signal was exclusively observed within the nucleus (Figure 4).

3.6. Analysis of Expression Patterns of CmSMXL6

qPCR was used to explore the expression profiles of CmSMXL6 in various organs and at different times for GR24 processing. As shown in Figure 5A, the CmSMXL6 gene was expressed in different tissues, with the highest relative expression level in the stem, approximately 19 times that in the terminal bud, and the lowest relative expression level in the terminal bud. Compared to the stem, the expression levels of CmSMXL6 were lower in both the roots and leaves.

An analysis of the expression levels of the CmSMXL6 gene in stems treated with GR24 revealed the upregulation of expression following treatment. The highest expression level was observed at 9 h post-treatment and was 16.86 times higher than that of the control group. Subsequently, the expression level began to decrease, reaching a level 8.83 times higher than that of the control at 12 h post-treatment (Figure 5B). Therefore, the induction of CmSMXL6 gene expression after exogenous GR24 treatment suggests its involvement in SLs signal transduction pathways in chrysanthemum.

3.7. The Heterologous Expression of CmSMXL6 Increased the Branching Number in A. thaliana

To further investigate the function of CmSMXL6, transgenic validation was performed in A. thaliana. Positive transgenic lines were screened using reverse transcription PCR (Figure 6A). The expression of CmSMXL6 in A. thaliana lines was examined via qPCR.

CmSMXL6 was not detected in wild-type A. thaliana, and lines 1 and 5 were selected for subsequent analysis because of the high expression levels of CmSMXL6 (Figure 6B). Phenotypic observations of transgenic lines revealed that the overexpression of CmSMXL6 significantly increased the branching number in A. thaliana but had a less pronounced effect in increasing the rosette leaf count (Figure 6C,D). A statistical analysis of the number of branches in A. thaliana revealed that in the overexpressing lines, there was a significant increase in the number of primary branches with five or more branches. Additionally, compared to the wild type, there was a noticeable decrease in the number of lines with two secondary branches and an increase in the number of lines with more than three secondary branches. (Figure 6E,F). These results indicate that CmSMXL6 was involved in regulating plant branching.

3.8. Analysis of Relative Expression Levels of Branching Genes in Transgenic A. thaliana

An analysis was conducted on the expression of SLs synthesis genes in transgenic lines. As depicted in Figure 7, the expression levels of the branch-suppressing genes AtMAX1, AtMAX2, AtMAX3, and AtBRC1 were all down-regulated to varying degrees in the transgenic lines. Notably, the down-regulation of AtMAX3 was the most significant, with its expression only 0.3–0.4 times that of the wild type. Conversely, the AtMAX4 gene in the strigolactones signaling pathway exhibited increased expression in the CmSMXL6 transgenic strain, while the expression level was 3.8–4.8 times higher than that of the wild type.

4. Discussion

The development of lateral branches in plants is regulated by endogenous hormones, external environmental factors, water availability, and nutrients. Plant hormones are considered crucial regulatory factors for plant growth and development, as they act as signaling molecules capable of inducing significant physiological effects even at low concentrations [25]. In this study, exogenous hormone treatment was applied to C. morifolium ‘Baltasar’. The results revealed that the growth of lateral buds was significantly promoted under 6-BA treatment. Conversely, GR24 and IAA treatments inhibited the growth of chrysanthemum lateral buds, consistent with findings in other species, such as peas, tobacco, and apples [29]. Furthermore, the growth rate of lateral buds varied under different hormone treatments. The lateral bud growth rate was highest under 6-BA treatment, consistent with the hormonal regulation of lateral bud growth. However, a decline in the lateral bud growth rate was observed on the 10th day under 6-BA treatment, possibly due to specific nutrient requirements for lateral bud development in this stage. Further experimental validation is necessary to determine the underlying cause. As research into branching development has advanced, it has become evident that plant branching is intricately governed by a complex network of hormones [30].

The expression of growth-related genes and hormone-related genes in lateral branches will undergo changes after hormone treatment. Inhibition by IAA coincided with a low dividing cell percentage and unchanged or increased expression of CmBRC1, CmDRM1, and CmPIN1 [31]. In our study, after the exogenous application of IAA, the expression levels of the growth-related genes CmBRC1 and CmDRM1, as well as the SLs-related genes CmMAX1 and CmSMXL6, were up-regulated to varying degrees in the treatment group compared to the control group. Conversely, the expression level of the cytokinin synthase gene CmIPT3 was down-regulated. The results indicate that IAA and SLs synergistically interact in promoting lateral shoot growth, with IAA facilitating the signal transduction of SLs while suppressing lateral branch development. The expression of PsIPT1 in the stems of SL mutants was significantly up-regulated, and the mutant showed increased sensitivity to exogenous cytokinin treatment in promoting lateral bud growth [32]. In this study, it was observed that the expression of the branching inhibitor gene CmBRC1 was significantly reduced after treatment with 6-BA. Additionally, the related expression levels of the SLs signaling pathway genes CmMAX1 and CmSMXL6 were up-regulated. These findings are consistent with results from studies in peas, indicating that SLs signal transduction is influenced by 6-BA. After treatment with GR24, the expression of CmPIN1 was significantly reduced, indicating that GR24 decreased the transport capacity of auxin. This suggests that SLs and IAA cooperatively control plant branching [5]. Furthermore, the relative expression level of CmIPT3 was notably decreased, suggesting that SLs may modulate the biosynthesis of cytokinins through feedback regulation, thereby exerting an inhibitory effect on branching [22]. These findings indicate that, in addition to regulating the expression of the key inhibitory branching gene CmBRC1 to control plant branching, these three hormones also mutually influence their synthesis- or transport-related genes, forming a complex network that ultimately impacts plant phenotypes.

D53/SMXLs act as inhibitors of the strigolactone signaling pathway, playing a crucial role in the regulation of downstream target genes of strigolactones [33]. This study cloned and functionally validated CmSMXL6 in chrysanthemum. A systematic evolutionary analysis revealed that CmSMXL6 is most closely related to homologous genes in pumpkin and small pecan and also shares a high degree of homology with AtSMXL6 in A. thaliana. The analysis of the expression patterns of CmSMXL6 in different tissues indicated its highest expression level in the stem, displaying tissue-specific expression, suggesting that CmSMXL6 may be associated with the growth of chrysanthemum lateral branches. Furthermore, it was observed that the expression of CmSMXL6 was up-regulated after GR24 treatment of chrysanthemum, indicating its responsiveness to SLs and its involvement in the signal transduction pathway of SLs. This finding is consistent with results obtained in A. thaliana and pea [34]. Previous research suggests that the SMXL6, SMXL7, and SMXL8 genes play a dual inhibitory role in the SL signaling pathway [18]. In this study, it was observed that the number of primary and secondary branches in these transgenic plants increased compared to the WT, and the relative expression level of AtBRC1 in the transgenic plants was down-regulated to varying degrees compared to the wild type. In A. thaliana, BRC1 is a gene involved in the suppression of branching; the down-regulation of AtBRC1’s relative expression levels suggests that it may be regulated by CmSMXL6, thereby promoting plant branching [13]. In addition, the SL signal is dependent on the expression of BRC1, as exogenous SL application fails to suppress the multi-branching phenotype in brc1 function-deficient mutants. Furthermore, the relative expression levels of the SL synthesis genes AtMAX1, AtMAX2, and AtMAX3 were down-regulated to varying degrees in CmSMXL6 transgenic lines. This is consistent with the observed multi-branching phenotype in A. thaliana and suggests that CmSMXL6 positively regulates plant branching by influencing the expression of the SL synthesis genes MAX1, MAX2, and MAX3 [35]. In this study, it was also observed that the relative expression level of AtMAX4 was up-regulated. This suggests that, due to the dual inhibitory function of SMXL6 in the SLs signaling pathway, it can inhibit downstream transcription factors while also repressing its own promoter. This forms a negative feedback regulatory system to maintain the stability of the SLs pathway. Therefore, it is speculated that this may be caused by changes in endogenous SLs hormone levels in plants. In conclusion, the levels of SLs, auxin, and CK may be controlled through feedback regulation, thereby influencing plant branching. However, further research is needed to elucidate the specific mechanisms involved.