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Article

The ClTFL1-ClGRFs Module Regulates Lateral Branch Number and Flowering Time via Auxin-Mediated Pathway in Watermelon (Citrullus lanatus)

by
Yaomiao Guo
1,2,†,
Yachen Liu
1,2,†,
Huanhuan Niu
1,2,
Yinping Wang
1,2,
Zihao Chen
1,2,
Jiaxin Cui
1,2,
Changbao Shen
1,2,
Shixiang Duan
1,2,
Qishuai Kang
1,2,
Huayu Zhu
1,2,
Sen Yang
1,2,
Dongming Liu
1,2,
Wenkai Yan
1,2,
Junling Dou
1,2,* and
Luming Yang
1,2,*
1
College of Horticulture, Henan Agricultural University, Zhengzhou 450046, China
2
Research Center of Cucurbit Germplasm Enhancement and Utilization of Henan Province, Zhengzhou 450046, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(9), 1022; https://doi.org/10.3390/horticulturae11091022
Submission received: 4 August 2025 / Revised: 22 August 2025 / Accepted: 25 August 2025 / Published: 1 September 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Versions Notes

Abstract

The early flowering and less lateral branches in watermelon hold significant agricultural value. The synergistic effects of these traits provide an ideal template for watermelon plant architecture improvement. However, the molecular regulatory networks underlying the development of lateral organs (including branches and flowers) in watermelon remain unclear. In this study, we found ClTFL1 knockout lines significantly promote flowering time and inhibit lateral branching and tendril formation, while also leading to a mild apical flower phenotype. These findings indicate that the function of ClTFL1 in watermelon is more extensive than that of its homologous genes in Arabidopsis, rice, and tomato. Through yeast two-hybrid screening, we identified the interacting proteins of ClTFL1, including members of the 14-3-3 family ClGRF8, ClGRF9, and ClGRF12. Bimolecular fluorescence complementation (BiFC) assays further demonstrated ClTFL1 could directly interact with the ClGRF8, ClGRF9, and ClGRF12 protein. The knockout of ClGRF8 and ClGRF12 leads to reduced lateral branches and early flowering. These phenotypes are highly consistent with those of ClTFL1 knockout mutants. Our findings demonstrate the important role of the ClTFL1-ClGRFs module in regulating lateral organ development and flowering time in watermelon, offering important targets for watermelon plant architectural modification and molecular breeding.

1. Introduction

The plant structure is determined by the meristematic activity of the shoot apical meristem (SAM) and axillary meristems (AMs) and is simultaneously regulated by the sequential development of lateral organs, including leaves, tendrils, branches, and flowers [1]. Watermelon (Citrullus lanatus) is a significant Cucurbitaceae crop, and it holds considerable importance in global horticulture [2]. Its plant architecture traits, especially flowering time and branching habits, directly affect cultivation efficiency, production costs, and final yield [3]. In commercial production, the ideal plant architecture should have two key characteristics: “early flowering” and “few lateral branches”. Early flowering can shorten the growth period, promote early fruit ripening, help plants avoid environmental stress at the end of the growing season, which achieves efficient crop rotation [4]. Less lateral branches can significantly reduce the complexity of the plant canopy, reduce the reliance on manual pruning, improve ventilation and light transmission conditions, and provide possibilities for dense planting and mechanized operations [5]. However, the current commercial watermelon varieties generally suffer from the problem of excessive lateral branches, with almost every node able to sprout branches [6], resulting in field cover, cumbersome management, significantly increased labor costs, and significantly limited yield per unit area due to the requirements of wide-row and wide-spacing planting.
Optimal timing of both the onset of reproductive development and the transition from branching to flowering within the inflorescence is particularly important for the reproductive success of flowering plants in response to seasonal cues [7,8,9,10,11]. For instance, in plants that flower only once, like Arabidopsis and most crops, an early transition to floral development accelerates the completion of the life cycle, which is advantageous in environments with short growing seasons [11,12,13]. However, precocious floral initiation can reduce fecundity and yield since floral formation occurs at the expense of lateral branch development, which supports the production of a greater number of flowers per plant. Conversely, delayed flowering formation increases branching and total flower potential but prolongs the time required for seed set [11,12,13,14,15]. So, there needs to be a balance between flowering formation and branching.
The flowering process represents the transition from vegetative to reproductive growth [4]. Mutations or altered expression levels of genes regulating this process can significantly alter flowering time, growth duration, and plant architecture in crops. FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) are two pivotal genes within the flowering pathway. Both belong to the evolutionarily highly conserved phosphatidylethanolamine-binding protein (PEBP) family, yet they exert antagonistic effects on flowering: FT promotes flowering while TFL1 suppresses it. FT and TFL1 determine floral initiation by competitively binding to the bZIP transcription factor FD or 14-3-3 proteins [15,16].
The TFL1 gene is expressed in the shoot apical meristem (SAM), where it prevents the premature transition from vegetative to reproductive growth. Mutations or down-regulation of TFL1 expression can convert indeterminate inflorescence to determinate ones and cause early flowering. More importantly, the role of TFL1 in maintaining indeterminate growth and repressing flowering is highly conserved across species [17,18,19,20,21,22]. Loss-of-function mutations in TFL1 homologs, such as the CENTRORADIALIS (CEN) gene in snapdragon, SELF-PRUNING (SP) in tomato, and CsTFL1 in cucumber, consistently lead to early flowering, determinate growth, and the formation of terminal flowers [17,18,19,20,21,22,23,24,25,26,27]. Besides its role in flowering regulation, additional functions of TFL1 had been progressively discovered. Recent studies indicated that the AtTFL1 gene regulates seed cavity size and the initiation timing of embryo expansion in Arabidopsis by interacting with ABI5 and RAN2, thereby regulating seed size [23]. Consequently, tfl1 mutant seeds were significantly larger than those of the wild type [23]. In barley, the loss-of-function mutant of HvCEN (a TFL1 homolog), besides exhibiting early flowering, displayed phenotypes including reduced spikelet number, fewer tillers, and lower yield [28]. In cotton, the TFL1 homolog GhSP played a crucial role in maintaining the indeterminate growth of both sympodial and monopodial branches. Mutations in GhSP resulted in the phenotypes of reduced lateral branches and early flowering [29,30].
While the conserved function of TFL1 suggests a critical role in watermelon, its specific mechanisms remain unexplored. We previously demonstrated that ClTFL1 regulates the number of lateral branches in watermelon through map-based cloning [21]. However, the molecular mechanism underlying ClTFL1 regulation of lateral branch development remains poorly understood. Furthermore, it is unclear whether ClTFL1 also regulates flowering time in watermelon, as observed in other species, or through which the ClTFL1 pathway modulates both lateral branching and flowering time in watermelon. These questions remain to be elucidated.
In this study, we demonstrated that ClTFL1 regulates both lateral branch development and flowering time in watermelon through gene-editing-based validation and construction of near-isogenic lines (NILs). Furthermore, to elucidate the underlying mechanism, we identified the interaction between ClTFL1 and three GRFs (ClGRF8, ClGRF9, ClGRF12) protein through yeast library screening and point-to-point verification. Gene editing of these ClGRFs (ClGRF8 and ClGRF12) confirmed that GRFs also regulate lateral branch development and flowering time in watermelon. Our work establishes the ClTFL1-GRFs module as a key regulator of plant architecture in watermelon, providing a crucial theoretical foundation for understanding flowering regulation and breeding for an ideal plant architecture in watermelon. It also offered new insights for improving cultivation practices and facilitating mechanized production in watermelon.

2. Materials and Methods

2.1. Plant Materials and Phenotype Measurement

The watermelon standard inbred line WT20 and its Cltfl gene near the isogenic line tfl1-NIL were both preserved in our laboratory [6]. The “YL” and “TC” high generation inbred lines of watermelon (C. lanatus) were collected from Yulin City and Tongchuan City, Shaanxi Province, China, respectively, for genetic transformation [3]. The plump seeds were selected and soaked in sterile water at 55 °C for 15–20 min. After the water was cooled to room temperature, soaking was continued for 2–4 h, and then the seeds were placed on moist gauze in a dark incubator at 30 °C to promote germination. After germination, the seedlings with roots were transplanted into seedling pots and subjected to a light cycle of 16 h of light and 8 h of darkness. The temperature was maintained at 25 °C during the day and dropped to 20 °C at night. When the seedlings grew 2–3 true leaves, they were transferred to a solar greenhouse for cultivation. Fertilization and pest control were carried out in accordance with standard operating procedures. All plants, including WT20, tfl1-NIL, wild-type (WT) controls (YL and TC), and all CRISPR/Cas9-mediated knockout mutant lines, were grown in greenhouses under natural light conditions in Zhengzhou, China. These experiments were conducted in the spring of 2023 and 2024 (April–June).
For experimental design and phenotype measurement, a completely randomized design (CRD) was conducted in this study. The number of lateral branches was recorded at 30 d after planting. Specifically, the lateral branch lengths were measured when they were longer than 1 cm. Each experiment underwent 3 biological replicates, each consisting of 10 plants of each genotype. The final sample size for statistical analysis was n = 30 for each genotype, as all planted plants have successfully grown and been included in the measurement.

2.2. Hormone Measurement

Hormone levels were measured by NJRuiYuan Co., Ltd. as described by Ruiz-Lozano [31]. The apices tissues of WT20 and tfl1-NIL were harvested and pooled from five independent plants to form one biological replicate, and three such biological replicates were analyzed per genotype. The apices of WT20 and tfl1-NIL (2 g) were ground into powder with liquid nitrogen. The powder was extracted with 0.5 mL 40% acetone (v/v), followed by vortexing for 2 min, then centrifugation at 8000× g for 5 min. The pellets were collected and extracted twice with 0.5 mL 50% acetone (v/v). The supernatant was purified with membrane filters (0.22 μm pore size). Hormone content was then measured by HPLC-MS/MS.

2.3. Transcriptome Analysis

The apices of WT20 and tfl1-NIL were used for RNA-seq analysis at 30 d after planting. Three biological replicates were performed for each sample. Total RNA was extracted and used for strand-specific RNA-Seq library construction. The RNA-seq libraries were sequenced on an Illumina HiSeqTM 4000 platform (Bai Mike, Beijing, China).
The obtained raw data were filtered and sequencing adapters were removed using Fastp (V0.23.4). The clean reads were used for alignment to the watermelon reference genome “97103” V2 using Hisat2 (V2.2.1), and the read numbers of annotated genes were counted by the feature Counts program. The data were further analyzed in R (V4.5.0), and the correlation between all samples using the cor function in R and visualized using the pheatmap package in R. The number of transcripts per million (TPM) reads for each gene was calculated based on the length of the gene and mapped read counts. Differential expression genes between WT20 and tfl1-NIL were identified using the DESeq2 package in R. A corrected p value of 0.05 was set as the threshold for DEG selection. The common differentially expressed genes in both comparison groups were selected for further analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of the common differentially expressed genes was performed using TBtools software (V2.056). The GO and KEGG enrichment results were visualized through the online website Weishengxin.

2.4. qRT-PCR Analysis

Total RNA was extracted from different organs (including roots, stems, leaves, flowers, ovaries, and apical and axillary apices) of watermelon WT20 and tfl1-NIL utilizing the Quick RNA isolation Kit (Huayueyang, Beijing, China). The reverse transcription was performed utilizing RNA of watermelon WT20 and tfl1-NIL as templates through HiScriptRII RT SuperMix for the qPCR (+gDNA wiper) Kit (Vazyme, Nanjing, China) to generate cDNA. qRT-PCR was conducted using the SYBR® Premix Ex Taq kit (Vazyme, China) and then carried out on the qPCR instrument ABIPRISM 7500 (Applied Biosystems, Foster City, CA, USA). The thermal cycler setup for qRT PCR was as described by Zhu et al. [32]. The watermelon ClActin gene was used as a control. Quantitative algorithms were used as described by Duan et al. [33]. All samples were subjected to three biological and technical replicates.

2.5. Yeast Two-Hybrid Assay

The full-length coding sequences (CDSs) of ClTFL1, ClGRF8, ClGRF9, and ClGRF12 were cloned to construct the pGBKT7 bait plasmid and pGADT7 prey plasmid for yeast two-hybrid (Y2H) assays. The yeast transformation method was used as described by Yang et al. [34]. Primer information is provided in Table S2. After verifying the absence of autoactivation, the bait and prey plasmids were co-transformed into yeast strain AH109. The transformed yeast cells were grown on SD-Trp-Leu medium plates at 30 °C for 2–4 d. Single yeast transformants were selected and transferred to SD-Trp-Leu-His-Ade/X-α-gal plates for an additional 2–4 d to test for protein–protein interactions. Colonies showing normal growth and turning blue were considered positive for interaction.

2.6. Bimolecular Fluorescence Complementation (BIFC) Assay

The CDSs of ClTFL1, ClGRF8, ClGRF9, and ClGRF12 were cloned and inserted into the Xba1 and BamH1 sites of the pSPYNE and pSPYCE vector [35], respectively. The recombinant plasmids were extracted and transformed into Agrobacterium GV3101(pSoup), and co-expression was observed in tobacco leaves. After 24 h of dark treatment followed by 48 h of light exposure, fluorescent signals were observed by a confocal microscope (Leica Microsystems, Wetzlar, Germany).

2.7. Watermelon Transformation

The knockout lines of ClTFL1, ClGRF8, ClGRF9, and ClGRF12 in watermelon were generated using the CRISPR/Cas9 system. Two single-guide RNA (sgRNA) binding sites in the CDS of ClTFL1, ClGRF8, ClGRF9, and ClGRF12 were designed by CRISPR design software (https://crispr.dbcls.jp/, accessed on 6 October 2022), respectively. The CRISPR/Cas9 vector construction and transformation were carried out following previously described methods [3]. The transformation assay was conducted using watermelon inbred lines YL and TC.

2.8. Statistical Analysis

Data graphing and analysis were carried out by GraphPad Prism software (V10), and significance analysis was conducted with SPSS 23.0 and GraphPad Prism software (V10). Each experiment was performed in triplicate. Statistical parameters and significance levels are noted in the figures, with asterisks indicating the results of statistical significance tests compared to the respective controls. For comparisons between two groups (WT20 vs. tfl1-NIL), the significant differences were tested using the Student’s t-test. For comparisons among more than two groups (e.g., WT vs. ClTFL1CR-1 vs. ClTFL1CR-2), one-way analysis of variance (ANOVA) was performed, followed by Duncan’s test for multiple comparisons, and differences were considered significant at the 0.05 and 0.01 levels. * indicates p < 0.05, while ** indicates p < 0.01. All primer information is listed in Table S2.

3. Results

3.1. ClTFL1 Regulates Flowering Time and Number of Lateral Branches in Watermelon

ClTFL1 was involved in the development of lateral branches in watermelon in our previous study [5], which was further validated by generating a near-isogenic line (NIL) of Cltfl (named tfl1-NIL) in the recurrent parent background WT20 [6]. Interestingly, phenotype observations revealed that the tfl1-NIL exhibited early flowering compared to its recurrent parent WT20 (Figure 1A).
The wild-type WT20 produced the first male flower at 24 d after transplanting, whereas the tfl1-NIL produced the first male flower as early as 15 d after transplanting (Figure 1B–D). Furthermore, the average flowering time of the tfl1-NIL female flowers was 13 d earlier than that of the WT20 (Figure 1E–G). More importantly, tfl1-NIL only showed a small number of lateral branches (three to five) during the early growth period, and no stagnant lateral branches or tendrils were formed in the remaining internodes (Figure 1J). Meanwhile, tfl1-NIL exhibited a mild flower topping phenomenon during the later stages of growth (Figure 1H,I). Collectively, these findings demonstrate that ClTFL1 was a negative regulator of flowering time and positive regulator of lateral branching.

3.2. Function Validation of ClTFL1 in Watermelon

Through genetic transformation, two ClTFL1 knockout lines were obtained in the background of “YL” (the wild-type watermelon), including ClTFL1CR-1 and ClTFL1CR-2 carrying 6 bp and 1 bp homozygous deletion alleles, respectively (Figure 2A). Phenotype observation revealed that in vegetative growth, all knockout mutants completely lacked tendrils and lateral branches at leaf axils above the fifth node, which was in sharp contrast to the wild-type plants (Figure 2B). In reproductive growth, all knockout mutant lines showed a much earlier opening time for both male and female flowers compared to the WT plants, showing an early flowering phenotype (Figure 2C–J). The number of lateral branches was significantly reduced compared to the wild-type plants (Figure 2N). It is worth noting that consistent with the tfl1-NIL, ClTFL1CR-1 and ClTFL1CR-2 knockout mutants exhibit a mild flower topping phenomenon in the later stage of growth (Figure 2K–M).

3.3. ClTFL1 Participates in Auxin-Mediated Pathway to Regulate the Flowering Time and the Number of Lateral Branches

To clarify the regulatory network of ClTFL1 in flowering time and lateral branching in watermelon, we conducted RNA-seq analysis. Correlation analysis of these samples showed high consistency among three biological replicates for each group, indicating that the RNA-seq data were reliable (Figure 3A). Compared to WT20, there were 1958 differentially expressed genes (DEGs) detected, with 1380 down-regulated and 578 up-regulated in tfl1-NIL (Figure 3B).
KEGG analysis revealed that most DEGs were enriched in pathways associated with photosynthesis-antenna proteins, metabolism, and zeatin biosynthesis (Figure 3C). GO enrichment analysis indicated that the DEGs participated in response hormones, the auxin-activated signaling pathway, and the response to cytokinin, particularly for the hormone-mediated signaling pathway (Figure 3D). Our RNA-Seq identified many DEGs associated with the auxin pathway (Figure 3D). Among these, 10 DEGs including ClGRF12, ClARF9, ClARF10, and ClIAA19 were selected for subsequent qRT-PCR validation in WT20 and tfl1-NIL (Figure 3E). A consistent expression pattern of these DEGs was observed in RNA-Seq and qRT-PCR analysis (Figure 3F). Auxin, as a key hormone regulating plant development, plays a central role in determining flowering time and lateral branch morphogenesis [36,37,38]. The tfl1-NIL exhibited an earlier flowering and less lateral branches compared with WT20, indicating that the tfl1-NIL might accumulate less auxin content. Accordingly, the endogenous hormone (IAA, zeaxanthin, JAMe, GA3) contents were measured between WT20 and tfl1-NIL. The endogenous auxin content of tfl1-NIL was significantly lower than that in WT20 (Figure S1), indicating that ClTFL1 likely functions as a negative regulator of auxin accumulation to affect plant development. Taken together, these findings indicate that ClTFL1 participates in auxin-mediated processes to regulate the flowering time and the number of lateral branches, providing a plausible mechanistic link between ClTFL1-mediated developmental changes and auxin regulation.

3.4. ClTFL1 Interacts with ClGRF8, ClGRF9, and ClGRF12

To investigate the potential mechanism of ClTFL1 in regulating flowering time and lateral branch development in watermelon, a yeast two-hybrid screen was employed against the cDNA library of the watermelon apical bud cDNA library. Multiple possible interacting proteins were successfully isolated using ClTFL1 as bait protein, including ClGRF8 (Cla97C08G154920.1, 14-3-3 protein), ClGRF9 (Cla97C07G131140.1, 14-3-3 protein), and ClGRF12 (Cla97C08G154060.1, 14-3-3 protein) (Figure 4A). The functions of these proteins in watermelon have not been validated yet. The results of the yeast two-hybrid assay indicated that ClTFL1 could interact with ClGRF8, ClGRF9, and ClGRF12, and the corresponding yeast showed blue staining under X-α-gal actosidase induction (Figure 4A). The bimolecular fluorescence complementation (BiFC) assays further validated the interactions between ClTFL1 and these proteins (ClGRF8, ClGRF9, and ClGRF12) in tobacco leaves. When infected with tumefaciens carrying plasmid combinations of ClTFL1-ClGRF8, ClTFL1-ClGRF9, and ClTFL1-ClGRF12, strong fluorescent signals were detected in tobacco leaves, while no signals were observed in the negative control group (Figure 4B). Furthermore, the analysis of cis-acting elements in the promoter sequences of the ClTFL1 and three 14-3-3 proteins (ClGRF8, ClGRF9, and ClGRF12) revealed that they all contained many similar regulatory elements, such as motifs responsive to meristem expression (CAT-box), gibberellin (P-box, TCCC-motif), auxin (TGA-element), abscisic acid (ABRE), and light signals (MRE, G-box, chs-CMA1a, 3-AF1 binding site, Box 4, GT1-motif, TCT-motif, AE-box, ATC-motif, GATA-motif) (Table S1). These results further suggested that ClTFL1 may regulate the initiation of tendrils, lateral branch development, and flower development processes by forming complexes with ClGRF8, ClGRF9, and ClGRF12.

3.5. Knockout of ClGRF12 Can Lead to Earlier Flowering Time

To investigate the function of ClGRF12 in watermelon, two knockout mutants were generated using the CRISPR/Cas9 system. Two independent knockout mutant lines, ClGRF12CR-1 and ClGRF12CR-2, were obtained, carrying homozygous deletions of 150 bp and 14 bp, respectively (Figure 5A). Both knockout mutant lines exhibited significantly earlier flowering compared to WT plants (Figure 5B–G). Specifically, WT plants began to produce male flowers at 30 d after transplanting, whereas ClGRF12CR-1 and ClGRF12CR-2 mutants produced male flowers at 24 d after transplanting (Figure 5C–E,I). A similar phenomenon was observed in female flowering, while WT plants formed their first female flowers at 39 d after transplanting, ClGRF12CR-1 and ClGRF12CR-2 mutants flowered at 32 d after transplanting (Figure 5F–H,J). Collectively, these results demonstrate that ClGRF12 plays a crucial role in regulating flowering time in watermelon.

3.6. Knockout of ClGRF8 Can Lead to Earlier Flowering Time and Reduced Number of Lateral Branches

To study the function of ClGRF8 in watermelon, we used the CRISPR/Cas9 system to design two targets at the first exon of the ClGRF8 gene and successfully obtained two independent knockout mutant lines (Figure 6A). Sequencing analysis showed that compared with the wild-type, ClGRF8CR-1, and ClGRF8CR-2 had deletions of 56 bp and 59 bp, respectively (Figure 6A). Phenotype observation showed that both knockout mutants exhibited characteristics of early flowering and reduced number of lateral branches (Figure 6B–K). These data indicate that the ClGRF8 protein is involved in the morphogenesis of watermelon flowers and the development of lateral organs.

4. Discussion

Plant architecture optimization is critical for the modern horticultural industry, where traits like reduced lateral branching and early flowering offer substantial agronomic advantages [5]. In watermelon, excessive lateral branching leads to sprawling vines with long internodes, resulting in inefficient land use and increased labor inputs for pruning, pest management, and harvesting [6]. Compact phenotypes with restricted branching enable high-density planting systems, improving resource utilization and reducing production costs. Concurrently, early flowering shortens the growth cycle, facilitating earlier fruit harvest and improved crop turnover—a key feature for climate-resilient and market-driven cultivation. The synergistic effects of these traits are particularly valuable for mechanized and labor-saving production systems. Thus, elucidating the genetic regulators of branching and flowering time is essential for breeding next-generation watermelon varieties suited for simplified, intensive, and sustainable agriculture.
TFL1 belongs to the FT/TFL1 gene family, which encodes a phosphatidylethanolamine binding protein (PEBP). Phosphatidylethanolamine, a major phospholipid in plant biofilms, plays a crucial role in protein recognition and signal transduction [39]. Additionally, TFL1 has been confirmed to be a key regulator in suppressing flowering and maintaining the infinite growth state in various plants, including Arabidopsis, tomato, and rice [39,40,41,42]. Our study confirms that ClTFL1 retains this conserved function in watermelon; specifically, ClTFL1CR-1 and ClTFL1CR-2 knockout mutants and tfl1-NIL exhibited early flowering and a mild terminal flower phenotype (Figure 1 and Figure 2). These observations align with previous reports in other crops, where TFL1 loss-of-function leads to early floral transition and determinate growth [41,42]. However, in our study, knockout of ClTFL1 in watermelon resulted in the loss of lateral branches and tendrils at the leaf axils of five or more nodes, with only a small number of branches remaining only at nodes three to five (Figure 2). This is significantly different from observations in Arabidopsis and tomato [40,41,42], indicating that watermelon ClTFL1 seems to play a broader role in controlling the initiation of lateral organs, including tendrils and branches. This functional expansion may reflect the unique developmental requirements of cucurbits, which rely on extensive lateral growth for vine elongation and fruit production.
Auxin is a central regulator of shoot branching and floral transition, acting through polar transport and signaling pathways [37,38]. The Arabidopsis BRC1 gene, which encodes a TCP transcription factor closely related to teosinte branched1 (tb1) in maize, is expressed in buds and can inhibit bud development, thereby suppressing lateral branch growth [43]. Additionally, BRC1 is essential for auxin-induced apical dominance [43]. Tomato’s SELF-PRUNING (SP) gene mutation alters auxin polar transport and response mechanisms, such as gravity-dependent bending and hypocotyl elongation, and this mutation also causes changes in free auxin levels and alterations in auxin-related gene expression patterns [44]. SP regulates tomato growth habits by influencing auxin transport and response [44]. These findings provide new insights into plant growth regulation and enhance crop productivity. Our transcriptome analysis revealed that a number of IAA-related DEGs were enriched, and auxin-related genes were down-regulated in tfl1-NIL compared with WT20, including ClARF9, ClARF10, and ClGRF12 (Figure 3E,F). Consistent with this, hormonal measurement demonstrated that tfl1-NIL accumulates a lower endogenous auxin (IAA) level compared to WT20 (Figure S1). These findings reveal that ClTFL1 influences auxin-mediated pathways; thus, we propose that ClTFL1 modulates auxin accumulation or signaling to control lateral organ development.
The 14-3-3 proteins are ubiquitous regulators of plant growth, functioning as scaffolds that modulate protein–protein interactions in response to auxin (IAA) and environmental cues [45]. This regulatory mechanism resembles the TFL1-FD-14-3-3 module described in Arabidopsis and rice, where TFL1 competes with FT-like proteins to bind 14-3-3 (GRF) proteins and FD transcription factors, forming a florigen repression complex (FRC) that inhibits flowering [46]. Our yeast two-hybrid and BiFC assays confirmed that ClTFL1 physically interacts with ClGRF8, ClGRF9, and ClGRF12 proteins (Figure 4), suggesting a conserved regulatory network in watermelon. Intriguingly, ClGRF8 and ClGRF12 appear to act as positive regulators of branching and flowering delay, as their loss led to fewer branches and early flowering (Figure 5 and Figure 6). And in future research, we will knock out ClGRF9 in watermelon and analyze its phenotypic effects on lateral branches and flowering time, which will clarify whether different 14-3-3 (GRFs) proteins have different or overlapping roles. In addition, future research should explore whether other factors compete with ClGRF8/12 protein for ClTFL1 binding, thereby regulating branch initiation.
Despite these advances, the functional specialization of 14-3-3 proteins in watermelon, particularly their roles in lateral organ development, has not been systematically characterized. In this study, we investigated the functions of ClGRF8 and ClGRF12 genes using CRISPR/Cas9 gene editing technology in watermelon. The result exhibited showed that the two genes play important roles in branching and flower regulation, knocking out ClGRF8 leads to a decrease in branching, while knocking out ClGRF12 exhibits an early flowering phenotype (Figure 5 and Figure 6). It is worth noting that these phenotype features are highly similar to the performance of ClTFL1 knockout lines. The yeast two-hybrid and bimolecular fluorescence complementation experiments further confirmed that ClTFL1 can directly interact with ClGRF8, ClGRF9, and ClGRF12 proteins (Figure 4), indicating that ClTFL1 functions by forming complexes with ClGRF8, ClGRF9, and ClGRF12 proteins. These findings confirm that ClTFL1 regulates the initiation of lateral branches in watermelon, not just the formation of flowers. Our findings suggest that modulating ClTFL1 or its 14-3-3 interactors could offer a genetic solution to reduce branching while maintaining desirable flowering timing. For example, using a weak effect ClTFL1 allele (such as tfl1-NIL) for variety improvement can reduce branching and pruning requirements while maintaining an ideal flowering time. In addition, through precisely editing the ClGRF8 or ClGRF12 genes, fine-tuning of branching traits can be achieved to avoid pleiotropy effects on flowering time. Furthermore, based on the identified auxin regulatory network, the development of hormone regulation strategies, such as targeted auxin transport inhibitors or tissue-specific auxin biosynthesis regulators, provides new avenues for plant configuration optimization. These findings provide an important theoretical basis and technical support for molecular design, breeding, and cultivation management of watermelons.
Based on this evidence, we proposed a model where the ClTFL1-ClGRFs module regulates both lateral branch development and flowering time via the auxin pathway in watermelon. Our research results indicate that ClTFL1 achieves a functional extension in watermelon and regulates the morphological characteristics and developmental processes of all lateral organs, including lateral branches, tendrils, and flowers. The main mechanism by which ClTFL1 functions includes the following: ClTFL1 directly interacts with ClGRF8 and ClGRF12 proteins to regulate lateral branch morphology and flower development. This model will deepen our understanding of shoot architecture plasticity in cucurbits and facilitate the development of new watermelon varieties optimized for high-density, mechanized cultivation.

5. Conclusions

In this study, we demonstrate that ClTFL1 plays a dual role in regulating both reproductive transition and vegetative architecture in watermelon. Concretely, ClTFL1 knockout mutants exhibited early flowering and significantly reduced lateral branching and tendril formation, revealing its novel function beyond floral repression. In addition, protein interaction studies demonstrated that ClTFL1 physically interacts with 14-3-3 proteins ClGRF8, ClGRF9, and ClGRF12, forming a key regulatory module. Functional analysis of these interactors showed that knockout of ClGRF8 reduces branching and causes early flowering; while knockout of ClGRF12 causes early flowering (Figure 5 and Figure 6), phenocopying aspects of the ClTFL1 mutants are observed, indicating that different 14-3-3 isoforms may compete to mediate the pleiotropic outputs of ClTFL1 signaling. Finally, we established a link to the auxin pathway, as the tfl1-NIL exhibited down-regulated expression of auxin-related genes and reduced endogenous IAA levels. Therefore, we conclude that the ClTFL1-ClGRFs module coordinately controls watermelon plant architecture by integrating branching and flowering signals, primarily through modulating the auxin pathway. These findings provide crucial targets for molecular breeding of improved watermelon varieties with optimized plant architecture.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11091022/s1, Figure S1: The contents of endogenous IAA, Zeatin, MeJA, and GA3 in WT20 and tfl1-NIL. Values are the mean of three biological replicates. ** represents p < 0.01 (Student’s t-test); Table S1: Cis-acting elements on the promoter of ClTFL1 and Cl14-3-3s (ClGRF8, ClGRF9 and ClGRF12); Table S2: The primer sequences in this study.

Author Contributions

L.Y. and J.D. designed the research; Y.G. and S.D. conducted the research; Y.L., Q.K., Y.W., Z.C., J.C. and C.S. contributed to experimental design and data interpretation; Y.G., J.D., D.L., H.N., W.Y., S.Y. and H.Z. edited the manuscript; L.Y., H.Z. and S.D. participated in data analysis and manuscript writing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (Grant No. 32172602, 3217180560, 32472739), Excellent Youth Foundation of Henan Scientific Committee (Grant No. 242300421030), and Science and Technology Project of the Tibetan Plateau Seed Breeding Technology Innovation Center (No. LSQSCNYQ2025006).

Data Availability Statement

Data generated in this study are deposited in the NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra, accessed on 8 September 2022) under accession number PRJNA1308744.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
BPBiological Process
CCCellular Component
MFMolecular Function

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Figure 1. Phenotypic characteristics of WT20 and tfl1-NIL. (A) Whole-plant morphology of WT20 and tfl1-NIL at 30 d. Scale bars, 10 cm. (B,C) Male flowers of WT20 and tfl1-NIL, respectively. (D) Quantitative statistics of the male flower (DMF) in WT20 and tfl1-NIL. (E,F) Female flowers of WT20 and tfl1-NIL, respectively. (G) Quantitative statistics of the female flower (DFF) in WT20 and tfl1-NIL. (H,I) Terminal buds of WT20 and tfl1-NIL, respectively. (J) Quantitative statistics of the number of lateral branches in WT20 and tfl1-NIL. ** significant at p < 0.01.
Figure 1. Phenotypic characteristics of WT20 and tfl1-NIL. (A) Whole-plant morphology of WT20 and tfl1-NIL at 30 d. Scale bars, 10 cm. (B,C) Male flowers of WT20 and tfl1-NIL, respectively. (D) Quantitative statistics of the male flower (DMF) in WT20 and tfl1-NIL. (E,F) Female flowers of WT20 and tfl1-NIL, respectively. (G) Quantitative statistics of the female flower (DFF) in WT20 and tfl1-NIL. (H,I) Terminal buds of WT20 and tfl1-NIL, respectively. (J) Quantitative statistics of the number of lateral branches in WT20 and tfl1-NIL. ** significant at p < 0.01.
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Figure 2. Phenotypic characteristics of WT, ClTFL1CR-1, and ClTFL1CR-2. (A) Mutation types of ClTFL1 knockout mutant lines. (B) Whole-plant morphology of WT, ClTFL1CR-1, and ClTFL1CR-2 at 30 d. Scale bars, 10 cm. (C–E) Male flowers of WT, ClTFL1CR-1, and ClTFL1CR-2, respectively. (F) Quantitative statistics of the male flower (DMF) in WT, ClTFL1CR-1, and ClTFL1CR-2. (G–I) Female flowers of WT, ClTFL1CR-1, and ClTFL1CR-2, respectively. (J) Quantitative statistics of the female flower (DFF) in WT, ClTFL1CR-1, and ClTFL1CR-2. (K–M) Terminal buds of WT, ClTFL1CR-1, and ClTFL1CR-2, respectively. (N) Quantitative statistics of the number of lateral branches in WT, ClTFL1CR-1, and ClTFL1CR-2. In (F,J,N), comparisons among WT, ClTFL1CR-1, and ClTFL1CR-2 are shown; one-way analysis of variance (ANOVA) was performed, followed by Duncan’s test for multiple comparisons, and differences were considered significant at the 0.01 levels. ** indicates p < 0.01.
Figure 2. Phenotypic characteristics of WT, ClTFL1CR-1, and ClTFL1CR-2. (A) Mutation types of ClTFL1 knockout mutant lines. (B) Whole-plant morphology of WT, ClTFL1CR-1, and ClTFL1CR-2 at 30 d. Scale bars, 10 cm. (C–E) Male flowers of WT, ClTFL1CR-1, and ClTFL1CR-2, respectively. (F) Quantitative statistics of the male flower (DMF) in WT, ClTFL1CR-1, and ClTFL1CR-2. (G–I) Female flowers of WT, ClTFL1CR-1, and ClTFL1CR-2, respectively. (J) Quantitative statistics of the female flower (DFF) in WT, ClTFL1CR-1, and ClTFL1CR-2. (K–M) Terminal buds of WT, ClTFL1CR-1, and ClTFL1CR-2, respectively. (N) Quantitative statistics of the number of lateral branches in WT, ClTFL1CR-1, and ClTFL1CR-2. In (F,J,N), comparisons among WT, ClTFL1CR-1, and ClTFL1CR-2 are shown; one-way analysis of variance (ANOVA) was performed, followed by Duncan’s test for multiple comparisons, and differences were considered significant at the 0.01 levels. ** indicates p < 0.01.
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Figure 3. RNA-seq analysis about the apex of WT20 and tfl1-NIL. (A) Heat map of Pearson correlation coefficients of RNA-seq data between WT20 and tfl1-NIL. (B) The number of genes that are differentially expressed between WT20 and tfl1-NIL. (C) KEGG enrichment analysis of cDEGs between WT20 and tfl1-NIL. (D) GO analysis of cDEGs between WT20 and tfl1-NIL. (E) Heat maps of auxin-related genes that were differentially expressed in apex of WT20 and tfl1-NIL. (F) The validation of auxin-related DEGs by qRT-PCR between WT20 and tfl1-NIL. Comparisons between two groups in each gene (WT20 vs. tfl1-NIL); the significant differences were tested using Student’s t-test. ** represents p < 0.01.
Figure 3. RNA-seq analysis about the apex of WT20 and tfl1-NIL. (A) Heat map of Pearson correlation coefficients of RNA-seq data between WT20 and tfl1-NIL. (B) The number of genes that are differentially expressed between WT20 and tfl1-NIL. (C) KEGG enrichment analysis of cDEGs between WT20 and tfl1-NIL. (D) GO analysis of cDEGs between WT20 and tfl1-NIL. (E) Heat maps of auxin-related genes that were differentially expressed in apex of WT20 and tfl1-NIL. (F) The validation of auxin-related DEGs by qRT-PCR between WT20 and tfl1-NIL. Comparisons between two groups in each gene (WT20 vs. tfl1-NIL); the significant differences were tested using Student’s t-test. ** represents p < 0.01.
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Figure 4. Protein interaction between TFL1 and GRFs protein. (A) Yeast two-hybrid (Y2H) of ClTFL1, ClGRF8, ClGRF9, and ClGRF12. The positive control was pGBKT7-53+pGADT7-T, while the negative control of ClGRF8, ClGRF9, and ClGRF12 protein were ClGRF8-AD+BK, ClGRF9-AD+BK, and ClGRF12-AD+BK, respectively. SD/-TL represents SD/-Trp/-Leu, SD/-THAL represents SD/-Trp/-His/-Ade/-Leu. (B) BiFC assays of ClTFL1, ClGRF8, ClGRF9, and ClGRF12. The negative control was nYFP+ClTFL1-cYFP. Scale bars, 50 μm.
Figure 4. Protein interaction between TFL1 and GRFs protein. (A) Yeast two-hybrid (Y2H) of ClTFL1, ClGRF8, ClGRF9, and ClGRF12. The positive control was pGBKT7-53+pGADT7-T, while the negative control of ClGRF8, ClGRF9, and ClGRF12 protein were ClGRF8-AD+BK, ClGRF9-AD+BK, and ClGRF12-AD+BK, respectively. SD/-TL represents SD/-Trp/-Leu, SD/-THAL represents SD/-Trp/-His/-Ade/-Leu. (B) BiFC assays of ClTFL1, ClGRF8, ClGRF9, and ClGRF12. The negative control was nYFP+ClTFL1-cYFP. Scale bars, 50 μm.
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Figure 5. Phenotypic characteristics of WT, ClGRF12CR-1, and ClGRF12CR-2. (A) Mutation types of ClGRF12 knockout mutant lines. (B) Whole-plant morphology of WT, ClGRF12CR-1, and ClGRF12CR-2 at 30 d. Scale bars, 10 cm. (C–E) Male flowers of WT, ClGRF12CR-1, and ClGRF12CR-2, respectively. (F–H) Female flowers of WT, ClGRF12CR-1, and ClGRF12CR-2, respectively. (I) Quantitative statistics of the d of female flower (DFF) in WT, ClGRF12CR-1, and ClGRF12CR-2. (J) Quantitative statistics of the d of male flower (DMF) in WT, ClGRF12CR-1, and ClGRF12CR-2. In (I,J), comparisons among WT, ClGRF12CR-1, and ClGRF12CR-2 are shown; one-way analysis of variance (ANOVA) was performed, followed by Duncan’s test for multiple comparisons, and differences were considered significant at the 0.01 levels. ** indicates p < 0.01.
Figure 5. Phenotypic characteristics of WT, ClGRF12CR-1, and ClGRF12CR-2. (A) Mutation types of ClGRF12 knockout mutant lines. (B) Whole-plant morphology of WT, ClGRF12CR-1, and ClGRF12CR-2 at 30 d. Scale bars, 10 cm. (C–E) Male flowers of WT, ClGRF12CR-1, and ClGRF12CR-2, respectively. (F–H) Female flowers of WT, ClGRF12CR-1, and ClGRF12CR-2, respectively. (I) Quantitative statistics of the d of female flower (DFF) in WT, ClGRF12CR-1, and ClGRF12CR-2. (J) Quantitative statistics of the d of male flower (DMF) in WT, ClGRF12CR-1, and ClGRF12CR-2. In (I,J), comparisons among WT, ClGRF12CR-1, and ClGRF12CR-2 are shown; one-way analysis of variance (ANOVA) was performed, followed by Duncan’s test for multiple comparisons, and differences were considered significant at the 0.01 levels. ** indicates p < 0.01.
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Figure 6. Phenotypic characteristics of WT, ClGRF8CR-1, and ClGRF8CR-2. (A) Mutation types of ClGRF8 knockout mutant lines. (B) Whole-plant morphology of WT, ClGRF8CR-1, and ClGRF8CR-2 at 30 d. Scale bars, 10 cm. (C,F,I) Male flowers of WT, ClGRF8CR-1, and ClGRF8CR-2, respectively. (E) Quantitative statistics of the d of female flower (DFF) in WT, ClGRF8CR-1, and ClGRF8CR-2. (D,G,J) Female flowers of WT, ClGRF8CR-1, and ClGRF8CR-2, respectively. (H) Quantitative statistics of the day of male flower (DMF) in WT, ClGRF8CR-1, and ClGRF8CR-2. (K) Quantitative statistics of the number of lateral branches in WT, ClGRF8CR-1, and ClGRF8CR-2. In (E,H,K), comparisons among WT, ClGRF8CR-1, and ClGRF8CR-2 are shown; one-way analysis of variance (ANOVA) was performed, followed by Duncan’s test for multiple comparisons, and differences were considered significant at the 0.01 levels. ** indicates p < 0.01.
Figure 6. Phenotypic characteristics of WT, ClGRF8CR-1, and ClGRF8CR-2. (A) Mutation types of ClGRF8 knockout mutant lines. (B) Whole-plant morphology of WT, ClGRF8CR-1, and ClGRF8CR-2 at 30 d. Scale bars, 10 cm. (C,F,I) Male flowers of WT, ClGRF8CR-1, and ClGRF8CR-2, respectively. (E) Quantitative statistics of the d of female flower (DFF) in WT, ClGRF8CR-1, and ClGRF8CR-2. (D,G,J) Female flowers of WT, ClGRF8CR-1, and ClGRF8CR-2, respectively. (H) Quantitative statistics of the day of male flower (DMF) in WT, ClGRF8CR-1, and ClGRF8CR-2. (K) Quantitative statistics of the number of lateral branches in WT, ClGRF8CR-1, and ClGRF8CR-2. In (E,H,K), comparisons among WT, ClGRF8CR-1, and ClGRF8CR-2 are shown; one-way analysis of variance (ANOVA) was performed, followed by Duncan’s test for multiple comparisons, and differences were considered significant at the 0.01 levels. ** indicates p < 0.01.
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Guo, Y.; Liu, Y.; Niu, H.; Wang, Y.; Chen, Z.; Cui, J.; Shen, C.; Duan, S.; Kang, Q.; Zhu, H.; et al. The ClTFL1-ClGRFs Module Regulates Lateral Branch Number and Flowering Time via Auxin-Mediated Pathway in Watermelon (Citrullus lanatus). Horticulturae 2025, 11, 1022. https://doi.org/10.3390/horticulturae11091022

AMA Style

Guo Y, Liu Y, Niu H, Wang Y, Chen Z, Cui J, Shen C, Duan S, Kang Q, Zhu H, et al. The ClTFL1-ClGRFs Module Regulates Lateral Branch Number and Flowering Time via Auxin-Mediated Pathway in Watermelon (Citrullus lanatus). Horticulturae. 2025; 11(9):1022. https://doi.org/10.3390/horticulturae11091022

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Guo, Yaomiao, Yachen Liu, Huanhuan Niu, Yinping Wang, Zihao Chen, Jiaxin Cui, Changbao Shen, Shixiang Duan, Qishuai Kang, Huayu Zhu, and et al. 2025. "The ClTFL1-ClGRFs Module Regulates Lateral Branch Number and Flowering Time via Auxin-Mediated Pathway in Watermelon (Citrullus lanatus)" Horticulturae 11, no. 9: 1022. https://doi.org/10.3390/horticulturae11091022

APA Style

Guo, Y., Liu, Y., Niu, H., Wang, Y., Chen, Z., Cui, J., Shen, C., Duan, S., Kang, Q., Zhu, H., Yang, S., Liu, D., Yan, W., Dou, J., & Yang, L. (2025). The ClTFL1-ClGRFs Module Regulates Lateral Branch Number and Flowering Time via Auxin-Mediated Pathway in Watermelon (Citrullus lanatus). Horticulturae, 11(9), 1022. https://doi.org/10.3390/horticulturae11091022

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