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. 2026 Jul 13;127(1):e71039. doi: 10.1111/tpj.71039

Branch architecture and flowering time are separately regulated by GhFL and GhSP2 in cotton

Zhenhui Guan 1,#, Qiuyan Cai 1,#, Pengfei Miao 1,2,#, Hailiang Cheng 1,3, Qiaolian Wang 1, Ji Liu 1,2,✉, Guoli Song 1,3,✉, Dongyun Zuo 1,✉
PMCID: PMC13358690  PMID: 42438919

SUMMARY

Crop flowering, a critical aspect of plant growth, is influenced by genetic and environmental factors. The indeterminate growth of cotton can lead to asynchronous flowering and boll setting. This study identified and characterized a determinate growth mutant, dt2, in Gossypium arboreum (G. arboreum) Shixiya 1 (SXY1) through EMS mutagenesis. The dt2 mutant exhibited main axis termination and shoot apical meristem (SAM) transition into flowers. Map‐based cloning revealed a single nucleotide mutation in the coding sequence (CDS) of GaFL (Ga07G0556), a FLORICAULA/LEAFY homolog responsible for the determinate growth phenotype. Virus‐induced gene silencing (VIGS) and protein interaction assays, including bimolecular fluorescence complementation, luciferase imaging, and GST pull‐down, were employed to functionally characterize GaFL and its Gossypium hirsutum (G. hirsutum) homolog GhFL (Gh_A07G051000). These integrated approaches demonstrated the critical roles of both proteins in cotton development. The results indicated that GhFL interacts with GhSP2 (Gh_D09G150100), both of which negatively influence flowering time in cotton. Furthermore, a reduction of GhSP2 expression resulted in increased GhJAZ5 (Gh_D06G087500) expression while overexpression of GhJAZ5 in cotton promoted early flowering. This research uncovers the genetic basis of determinate growth and provides insights into the mechanisms of flowering regulation in cotton. These findings have significant implications for breeding strategies to improve cotton plant architecture.

Keywords: cotton, determinate growth mutant, FLORICAULA/LEAFY HOMOLOG, flowering regulation mechanisms

Significance Statement

GhFL is capable of altering the growth pattern of the cotton shoot apex from indeterminate to determinate. Despite this, lateral branches of TRV: GhFL plants maintain indeterminate growth. However, the lateral branches of the mutant dt2, derived from FL, continued to exhibit indeterminate growth. Although the reduction in GhSP2 expression did not alter the indeterminate growth pattern of cotton, it facilitated flowering and decreased JA content in plants. This action consequently led to the up‐regulation of GhJAZ5 expression. The GhJAZ5 transgenic line exhibited significantly earlier flowering, indicating a potential linkage between JA signaling pathways and flowering regulation.

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INTRODUCTION

Cotton, a globally cultivated economic crop, serves as a significant source of natural fiber (Wang et al., 2019). Flowering and architectural patterns are intricately connected and play critical roles in mechanized crop management and harvesting practices (Huang et al., 2022). The distinct determinate and indeterminate growth habits of cotton plants profoundly influence their branching patterns and inflorescence density. Two naturally occurring mutants displaying determinate sympodial growth have been identified in allotetraploid cotton: the cluster branch (cl1) in Gossypium hirsutum (G. hirsutum) and the short branch (cl2) in Gossypium barbadense (G. barbadense) (Kearney, 1930; Pathak et al., 1975). Designated as nulliplex‐branch (nb) in both G. hirsutum and G. barbadense, these mutants have been widely utilized into breeding. The nulliplex‐branch phenotype is characterized by the absence of fruit branches, resulting in the direct attachment of one or more clustered flowers with elongated pedicels to the main stem. This plant architecture creates a compact growth habit that enables high‐density planting and thereby increases cotton yield (Silow, 1946; Wang et al., 2018). Although nb mutants have been widely used in breeding improvement, the genetic networks in coordinating flowering time and plant architecture remain largely unexplored. The mechanisms underlying monopodial and sympodial SAM development have been extensively documented in prior research. Monopodial growth, characterized by an indeterminate SAM, is primarily observed in main stems and vegetative branches. In contrast, sympodial growth, associated with a determinate SAM, is typically observed in fruiting branches. More specifically, determinate growth (sympodial) is known to delay flowering and facilitate the formation of fruiting branches, whereas indeterminate growth (monopodial‐like in nulliplex) promotes early flowering directly from axillary buds (Sun et al., 2016).

FLORICAULA/LEAFY (FL/LFY) is a transcription factor that regulates floral meristem organization and the transition from vegetative to reproductive growth (Blázquez et al., 1997). It facilitates flower formation by activating floral meristem identity genes, including APETALA 1 (AP1) (Jin et al., 2021). LFY facilitates floral meristem emergence (Liljegren et al., 1999; Weigel et al., 1992), triggers flowering onset (Mandel & Yanofsky, 1995), and prevents the reversion of floral meristems to a vegetative state (Mizukami & Ma, 1997). Nevertheless, functional studies of FL/LFY in cotton remain limited compared to those in other crops.

The phosphatidylethanolamine‐binding protein (PEBP) gene family, also known as CENTRORADIALIS/TERMINAL FLOWER 1/SELF‐PRUNING (CETS) (McGarry & Ayre, 2012), is essential for regulating plant architecture and flowering. TERMINAL FLOWER 1 (TFL1) acts as a signaling molecule, translocating from inner to outer cells in the inflorescence meristem (IM) (Conti & Bradley, 2007). During the flowering transition, TFL1 represses flowering‐related genes like LFY and AP1, maintaining indeterminate differentiation of IM (Goretti et al., 2020). In G. hirsutum, dynamic changes in the expression levels of GhSFT (SINGLE FLOWER TRUSS) and GhSP (SELF‐PRUNING, TFL1 orthologs) influence the differentiation direction of the SAM. Silencing of GhSP induced both monopodial and sympodial meristems to terminate precociously, whereas the two‐copy knockout of GhNB (Nulliplex‐branch, TFL1 orthologs) led to the termination of the main and lateral shoots in flowers, resulting in pronounced dwarfism in the plants (Chen et al., 2019; McGarry et al., 2016). Nonetheless, whether the interaction dynamics between FL/LFY and TFL1 could ultimately determine cotton plant architecture by balancing SAM maintenance with indeterminate growth remains unclear.

Beyond genetic regulation, the integration of environmental signals and plant hormones profoundly influences flowering. Jasmonates, including jasmonic acid (JA), methyl jasmonate (MeJA), and jasmonic acid isoleucine (JA‐Ile), have been demonstrated to play a critical role in regulating the transition from inflorescence meristem to floral meristem and in affecting floral organ development (Koo & Howe, 2012; Schaller & Stintzi, 2009; Wasternack & Hause, 2013; Wiszniewski et al., 2014). Jasmonate ZIM‐domain (JAZ) proteins in JA signaling pathway, regulate flowering time in Arabidopsis (Thatcher et al., 2016; Zhai et al., 2015), Solanum lycopersicum (Yu et al., 2018), and Chrysanthemum morifolium (Guan et al., 2021). However, research on the relationship between JAZ proteins and the regulation of flowering in cotton requires further investigation.

Cotton, which exhibits indeterminate growth, can be harvested within a single year through human domestication (Yuan et al., 2021). Growth habit is a significant factor that determines cotton production. Here, we obtained a determinate growth (dt2) mutant from the G. arboreum cultivar SXY1. Map‐based cloning revealed that dt2 is a novel mutant allele of the FL/LFY homolog, and its function was confirmed in both allotetraploid and diploid cotton through VIGS. The dt2 mutation terminated indeterminate growth of the main stem, while axillary buds maintained their differentiation potential. Interaction assays confirmed that GhFL directly interacts with GhSP2 (SELF‐PRUNING2, a TFL1 ortholog) in G. hirsutum. Further analysis showed that reduced GhSP2 expression altered jasmonate levels and upregulated GhJAZ5 expression and concurrently promoted flowering. Our findings provide insights into the mechanisms regulating cotton flowering. These results have significant implications for cotton flowering and offer new opportunities for breeding strategies to enhance yield.

RESULTS

Cloning the determinate growth 2 (dt2) gene in G. arboreum

A homozygous determinate growth 2 (dt2) mutant was obtained according to treatment of G. arboreum SXY1 with mutagen EMS. The average plant heights of SXY1 and dt2 plants were 91.6 ± 7.2 cm and 46.6 ± 3.0 cm, respectively (Figure 1a,d; Figure S1). The dt2 mutant displayed obvious determinate growth with a terminal boll at the top of the main stem (Figure 1b,c). However, axillary buds maintain differentiation capacity in both the wild‐type SXY1 and the dt2 mutant, exhibiting an indeterminate growth phenotype (Figure 1e,f).

Figure 1.

Figure 1

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The phenotype of determinate growth and indeterminate growth in dt1 and wild‐type Shixiya 1.

(a, d) The plant architecture of the dt2 mutant and the wild‐type Shixiya 1 at boll stage (Scale bar = 10 cm).

(b, c) The destiny of the SAM in the dt2 mutant and the wild‐type Shixiya 1. The white arrow indicates that the SAM became terminal boll in the dt2 mutant (c) (Scale bar = 3 cm) compared with the young leaf formed in the wild‐type Shixiya 1 (b) (Scale bar = 2 cm).

(e, f) The fruit branch of the dt2 mutant and the wild‐type Shixiya 1. The white arrow indicates that the axillary buds of the lateral branches became bolls (Scale bar = 5 cm).

To identify the causal locus for determinate growth of dt2, an indeterminate growth cultivar DQJ (Da Qing Ji) and the dt2 mutant were used as parents to construct an F2 population through hybridization and self‐fertilization. All F1 plants exhibited indeterminate growth. Among the F2 population, the segregation ratio of indeterminate growth and determinate growth was 3:1, χ 2 (1, 0.05) = 3.84 (Table S2). These data further indicate that a single recessive mutation controls the determinate growth trait in dt2. The bulk segregation analysis (BSA) revealed that the Δsingle nucleotide polymorphism (ΔSNP) index, exceeding the 99% confidence threshold, was situated on chromosome A07 between 6 Mb and 11 Mb (Figure 2a) by aligning sequence reads to the reference SXY1 genome. Subsequently, we developed 14 kompetitive allele specific PCR (KASP) markers spanning the genomic region from 3 792 070 bp (SNP marker GH900019) to 6 594 050 bp (SNP marker GH900043) (Table S1). Utilizing these markers, we conducted genotype testing in 123 recessive lines of the F2 generation population and discovered that the determinate growth trait was tightly linked to the 5.86–6.00 Mb interval, specifically located within the 0.14 Mb genomic region between KASP markers SNP‐GH900040 (pos.5865894) and SNP‐GH900038 (pos.5996879) (Figure 2b), which contained 8 genes (Table S3). The nucleic acid sequences of the 8 candidate genes between the two parents were aligned and an SNP was detected in the first exon of Ga07G0556 open reading frame (ORF). Additionally, no nucleotide mutations were observed in the other candidate genes within the dt2 mutant (Figure S2). This SNP resulted in the initiation codon mutation from ATG to ATA in the dt2 mutant, potentially causing a frameshift and the deletion of 18 amino acids in the N‐terminal (Figure 2c; Figure S3). Ga07G0556 encodes a putative FL protein (Coen et al., 1990), which is related to plant flowering processes. We hypothesized that Ga07G0556 was the candidate gene in the dt2 mutant responsible for the determinate growth in G. arboreum. To verify this hypothesis, VIGS technology was performed to silence the GaFL gene in SXY1 background. The phenotype of the GaFL‐silenced lines was also similar to that of the dt2 mutant (Figure 3). These findings provided strong evidence that the identified GaFL candidate gene is responsible for the determinate growth phenotype.

Figure 2.

Figure 2

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Fine mapping and cloning of determinate growth 2.

(a) qtl analysis of the determinate growth phenotype in an F2 segregating population, the determinate growth locus was mapped on chromosome A07 from 6 Mb to 11 Mb. The red arrow indicates the only window with a ΔSNP value exceeding the 99% significance threshold confidence interval across the whole genome. The ΔSNP index (SNP index of the determinate growth bulk population subtracted from that of the indeterminate growth bulk population) and its 99% confidence interval are shown as black curves and red dotted lines (ΔSNP index >0.5), respectively.

(b) Genetic mapping of the determinate growth locus in a population with 123 F2 recessive individual plants by KASP. The number of recombinants was shown below the blue line.

(c) The initiation codon from ATG to ATA mutation in the first exon of Ga07G0556.

Figure 3.

Figure 3

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Functional verification of GaFL in Shixiya 1 and GhFL in TM‐1.

(a) The phenotype of TRV: GaFL plants.

(b) The phenotype of TRV: GhFL plants. The white arrow indicates that the shoot apical meristem became terminal boll in (a) TRV: GaFL or (b) TRV: GhFL plants. The red arrow indicates that axillary buds maintain differentiation capacity in both TRV: 00 and TRV: GaFL or TRV: GhFL plants.

(c) The level of GaFL transcript in the leaves of TRV: GaFL plants and TRV: 00 plants. Data represent the mean of three independent biological replicates ± SEM. Gene expression data were normalized to GaHIS3. **P < 0.01 (Student's t‐test).

(d) Relative expression of GhFL in TRV: GhFL and TRV: 00 plants. Data represent the average of three independent biological replicates ± SEM. Gene expression was normalized to GhHistone3. **P < 0.01 (Student's t‐test).

(e) Relative expression of GhFL in the shoots of TM‐1 from 1 TLS to 5 TLS, normalized to GhHistone3.

(f) Relative expression of GhFL in different tissues of TM‐1, normalized to GhHistone3. Error bars represent the mean ± SEM of three independent experiments. **P < 0.01 (Student's t‐test).

(g) Subcellular localization of GhFL protein (Scale bar = 50 μm).

The homolog of GaFL was also responsible for determinate growth in G. hirsutum

Although GaFL has been demonstrated to control determinate growth in G. arboreum, it remains unclear whether this mechanism is conserved across other Gossypium species, especially in G. hirsutum, a globally cultivated species whose genetic background and agronomic traits hold substantial practical significance. Chromosomal mapping identified two FL loci in G. hirsutum (AD1 genome) at chromosomes A07 (Gh_A07G051000) and D07 (Gh_D07G051800) (Figure S4a). Considering the diploid G. arboreum (AA genome), our analysis concentrated on the A07 chromosomal GhFL‐At (hereinafter referred to as GhFL) locus. GhFL, a homolog of GaFL in G. hirsutum, was identified via sequence alignment. GhFL is distinguished from GaFL by a single SNP in the CDS region (Figure S4d). To confirm the functions of GhFL, virus‐induced gene silencing (VIGS) assays were performed in the cultivar TM‐1 (Figure 3b). Quantitative reverse transcription PCR (qRT‐PCR) demonstrated 54.8% ± 5.2% and 52.6% ± 6.0% reduction in GhFL expression in TRV: GhFL‐1 and TRV: GhFL‐2 plants, respectively, relative to the TRV:00 empty vector control (P < 0.01, Student's t‐test), confirming effective mRNA degradation via VIGS (Figure 3d). Silencing of GhFL led to the differentiation of the SAM into flower buds and resulted in the termination of the main stem with the formation of flowers at the stem top, thus producing a terminal flower (Figure 3b). This phenotype recapitulates the key determinate growth characteristic observed in the TRV: GaFL plants (Figure 3a). Also, axillary buds maintained differentiation capacity in VIGS‐treated plants, resulting in indeterminate growth phenotype (Figure 3b). The results demonstrate that in both G. arboreum and G. hirsutum, the phenotype exhibits similar characteristics, including a determinate main stem and indeterminate axillary meristems.

To identify the expression pattern of GhFL, SAM samples were collected from the first to fifth true leaf stages (1–5 TLS) of the cultivar TM‐1. GhFL exhibited a gradual increase in expression levels from 1 TLS to 5 TLS (Figure 3e). Furthermore, the expression levels of GhFL were determined in various nutritional organs of G. hirsutum TM‐1, including roots, stems, leaves, and SAM, as well as its relative expression in floral organs such as petals, anthers, styles, and pollen. Results showed that GhFL expression was most pronounced in the SAM, followed by high levels in roots and stems, and weak expression in floral organs (Figure 3f). Additionally, the transient expression of GhFL‐GFP fusion protein in Nicotiana benthamiana (N. benthamiana) leaves showed that GhFL protein localized within the cell nucleus (Figure 3g), underscoring its potential regulatory function.

GhFL proteins interact with GhSP2, a key regulator in plant flowering

Previous work had shown that SP genes played a crucial role in orchestrating the architecture and flowering in cotton (Liu et al., 2023; McGarry et al., 2016; Prewitt et al., 2018; Si et al., 2018). All plants with silenced GhFL expression invariably developed terminal flowers, culminating in a determinate growth form (Figure S5). This observation suggests GhFL and GhSP (Gh_D07G116400) share a partially similar function (Liu et al., 2023, Si et al., 2018), hinting at potential interactive pathways influencing plant architecture. Based on these preliminary insights, we hypothesized an interaction between GhFL and GhSPs in G. hirsutum.

To explore this hypothesis, BiFC, LCI, and pull‐down assays were employed to demonstrate a physical interaction between GhFL and GhSP2 proteins. A pull‐down assay was also performed to identify the interaction in vitro, showing that GhFL fused with GST (GhFL‐GST) was able to interact with GhSP2 fused with GFP (GhSP2‐GFP) (Figure 4a). In cotton protoplasts, pronounced fluorescence signals were observed in the nucleus, indicating a significant interaction between GhFL and GhSP2 in vivo. Conversely, the negative control samples showed no fluorescence (Figure 4b). The LCI assay provided additional validation of the in vivo interaction between GhFL and GhSP2 (Figure 4c). These results collectively affirm the conjecture that GhFL interacts with the GhSP2 protein in vivo and in vitro. Whether GhFL additionally modulates GhSP2 expression at the transcriptional level remains an open question.

Figure 4.

Figure 4

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Protein interaction of GhFL and GhSP2.

(a) Pull‐down assay. Recombinant GST‐GhFL with GFP‐GhSP2 proteins was used for the pull‐down assay. IB, immunoblot.

(b) BiFC assay of protein interactions of GhFL and GhSP2 in G. hirsutum protoplasts. Scale bar = 20 μm.

(c) LCI assay of protein interactions of GhFL with GhSP2 in N. benthamiana.

Typically, flower bud differentiation in the early maturing cultivars begins at the third true leaf stage (3 TLS), while in the late‐maturing cultivar, it commences at the fifth true leaf stage (Cheng et al., 2021). GhSP2 continuously accumulates prior to the onset of flower bud differentiation in both cultivars, indicating a strong correlation between GhSP2 and flower bud differentiation (Figure 5a). To test whether GhSP2 also participates in the regulating of flower bud differentiation in cotton, VIGS was performed in the cultivar TM‐1(Figure 5b). A 55.8% ± 12.2% and 57.3% ± 6.8% reduction in GhSP2 expression was observed in TRV: GhSP2‐1 and TRV: GhSP2‐2 plants, respectively, relative to the TRV:00 empty vector control (P < 0.01, Student's t‐test) through qRT‐PCR. This confirmed effective mRNA degradation via VIGS and demonstrated that GhSP2 expression was significantly diminished in treated plants compared to the control (Figure 5c). Silencing of GhSP2 resulted in advanced flowering times and lower height, suggesting that GhSP2 could inhibit flowering and increase plant height (Figure 5d,e). However, the shoot apex of the TRV: GhSP2 lines exhibits indeterminate growth without terminal flower formation (Figure 6b), which differs from that of the TRV: GhFL lines, indicating different pathways mediated by GhFL in regulating flowering time and determinate growth in cotton. Additionally, GhSP2 was overexpressed in Arabidopsis to further confirm its functions in flowering. Overexpression of GhSP2 in Arabidopsis significantly delayed flowering (Figure S6a–c), increased rosette leaf number, and reduced primary rosette branches (Figure S6d,f), indicative of a prolonged vegetative phase. Abnormal inflorescence structures were also identified in the transgenic lines, where floral clusters were surrounded by whorled leaf‐like structures at the uppermost nodes. This alteration resulted in the abnormal morphology of the inflorescences and inhibited the completion of normal sexual reproduction (Figure S6g–j). The results of paraffin sections revealed that the floral organs of the transgenic lines lacked anthers, styles, pollen, and ovules (Figure S6k–n). In conclusion, overexpression of GhSP2 significantly delayed the flowering time and markedly perturbed the structure of inflorescences in Arabidopsis, resulting in abnormal phenotypes, such as the enlargement of the calyx and the formation of spherical protrusions at the center of the inflorescence.

Figure 5.

Figure 5

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Functional verification of GhSP2 in TM‐1.

(a) The expression pattern of GhSP2 at different stages in TM‐1 and ZM425. Gene expression was normalized to GhHistone3.

(b) The phenotype of TRV: GhSP2 plants. Scale bar = 15 cm.

(c) Relative expression levels of GhSP2 in TRV: 00 and TRV: GhSP2 plants. Gene expression was normalized to GhHistone3.

(d, e) Statistics on (d) plant height and (e) node of the first fruiting branch of TRV: 00 and TRV: GhSP2 plants. The error bars indicate mean ± SEM of three independent experiments. **P < 0.01, (Student's t‐test).

Figure 6.

Figure 6

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Functional verification of JAZ5 in YZ1.

(a) Difference in jasmonates levels in TRV: 00 and TRV: GhSP2 plants.

(b) Phenotype of GhJAZ5 in overexpression lines (OE) and RNA interference lines (RNAi) plants (Scale bar = 15 cm).

(c) Flowering time of YZ1, OE and RNAi plants. The error bars indicate mean ± SEM of three independent experiments. **P < 0.01, (Student's t‐test).

(d) Subcellular localization of GhJAZ5 protein (Scale bar = 50 μm).

GhSP2 mediated the JA content and altered the expression of JAZ regulating flowering in cotton

To further delineate the potential mechanism by which GhSP2 regulates flowering time, the hormone levels in the shoot apexes were detected. The results indicated that the levels of N‐[(−)‐Jasmonoyl] − (L)‐valine (JA‐Val), cis (+)‐12‐Oxophytodienoic acid (OPDA), and methyl jasmonate (MeJA)—key compounds in the jasmonic acid synthesis and signal transduction pathways—were elevated in plants with silenced GhSP2 (Figure 6a). Within the JA signaling pathway, JAZ proteins serve as negative regulators of JA signaling. Beyond its pivotal role in plant defense responses, JAZ extensively participates in regulating plant developmental processes. qRT‐PCR results showed that the expression of GhJAZ1‐GhJAZ5 was significantly upregulated in TRV: GhSP2 plants (Figure S7a).

Additionally, the expression patterns of five GhJAZs were analyzed. The five GhJAZs exhibited distinct expression patterns in various tissues, with GhJAZ5 showing higher expression in petals, anthers, pistils, and pollen (Figure S7b). These results implied that GhJAZ5 may function in the JA signaling transduction during the JA content regulation mediated by GhSP2 to control flowering time in cotton. To verify this hypothesis, GhJAZ5 transgenic (OE) and RNA interference (RNAi) lines were utilized, and qRT‐PCR experiments confirmed differences in GhJAZ5 expression levels among wild‐type, OE, and RNAi lines (Figure 6b and Figure S7c). Analysis of the flowering times across the three lines revealed that overexpression of GhJAZ5 prompted early flowering, whereas suppression of GhJAZ5 resulted in delayed flowering (Figure 6c). Furthermore, the GhJAZ5 protein exhibited subcellular localization within the nucleus (Figure 6d). Taken together, these results demonstrated that GhSP2 acted as a key regulator, mediated the JA contents and the GhJAZ5 expression adjusting the JA signaling pathway, controlling flowering time in cotton.

DISCUSSION

The role of FLORICAULA/LEAFY (FL/LFY) in plant flower differentiation and tissue formation is pivotal, particularly during the transition from the vegetative to the reproductive growth stages (Schultz & Haughn, 1991). To delineate the function of GhFL in cotton, we employed a VIGS assay and found that the GhFL silenced plants exhibited the self‐pruning phenomenon, which was accompanied with early flowering. In cotton production, artificial decapitation, a crucial process for yield improvement, is labor‐intensive and difficult for chemical manipulation (Liang et al., 2020). The determinate growth phenotype resulting from GhFL silencing mimics the physiological outcome of artificial decapitation. Research into the function of GhFL provides valuable insights into cotton decapitation technology, identifying potential targets for genetic manipulation to enhance cotton plant structure and achieve agricultural gains.

After GhFL was silenced, the growth of the main stem transitioned from indeterminate to determinate, whereas the lateral branches maintained their normal indeterminate development. Further investigation is necessary to ascertain whether GhFL impacts the whole plant or localized determinate growth. Additionally, the bracts of TRV: GhFL plants exhibit abnormal development, characterized by more pronounced bracts (Figure S5). This is analogous to the aberrant flower morphology phenotype induced by lfy mutations in Antirrhinum majus (Azpeitia et al., 2021), which suggests the potential involvement of GhFL in the development of floral morphology.

As demonstrated in Arabidopsis, LFY directly binds to a distal regulatory region (approximately 2.8 kb downstream of the transcription start site) within the TFL1 promoter to activate its expression (Serrano‐Mislata et al., 2017). Building on this conserved regulatory mechanism in flowering plants, we hypothesize that GhFL may exhibit a similar binding affinity for the GhSP2 promoter. This hypothesis, distinct from the experimentally validated GhFL‐GhSP2 protein interaction reported in this study, will be tested in subsequent studies using DNA affinity purification sequencing and chromatin immunoprecipitation‐qPCR assays.

Although TFL1 lacks transcription factor activity, it serves as a central integrator in flowering time regulation. In Arabidopsis, TFL1 inhibits the FT‐FD complex to prevent precocious flowering (Zhu et al., 2020). Consistent with this, ectopic expression of GhSP2 in Arabidopsis significantly delayed the floral transition, prolonging vegetative growth (Figure S6g–j). This developmental alteration consequently affected overall plant architecture and reproductive competence. Transgenic lines exhibited abnormal inflorescence morphology characterized by whorled leaf‐like structures enveloping floral clusters at apical nodes, ultimately impairing normal sexual reproduction.

These findings are consistent with those of Prewitt et al., who subsequently demonstrated using yeast two‐hybrid assays that GhSP2 (GhTFL1‐L1) binds GhFD proteins in vitro, supporting functional conservation between G. hirsutum and Arabidopsis (Prewitt et al., 2018). Further functional complementation assays revealed that among cotton TFL1‐like genes, only GhSP, GhTFL1‐L2, and GhBFT‐L2 partially rescued the early flowering phenotype of the Arabidopsis tfl1‐14 mutant. In contrast, GhTFL1‐L1 (GhSP2) failed to complement the mutation, indicating functional divergence between GhSP2 and other TFL1‐like orthologs. This suggests that GhSP2 may not participate directly in maintaining indeterminacy in inflorescence meristems.

In species such as tomato, the sp mutation converts plant growth from indeterminate to determinate, leading to synchronized flowering and maturation (Pnueli et al., 1998). Similarly, in cotton, GhSP has been shown to modulate growth habit, while GhSP2 primarily influences flowering time. Phylogenetic analysis indicates that GhSP is closely related to CEN genes from Arabidopsis (AtATC) and Antirrhinum majus (AmCEN), whereas GhSP2 clusters with TFL1 (Figure S8). Although both genes belong to the PEBP family, they appear to regulate distinct aspects of development. GhSP primarily regulates growth habit, whereas GhSP2 specifically influences flowering time. Whether this functional specialization occurs through separate signaling pathways remains unclear. Therefore, we speculate that GhSP and GhSP2 may have different mechanisms regulating flowering.

JA serves as both a potent inhibitor of vegetative growth and a positive regulator of reproductive and defense mechanisms (Chung & Howe, 2009). In our research, the reduction of GhSP2 expression affected JA levels in cotton and upregulated the expression level of GhJAZ5. Silencing GhSP2 or overexpression of GhJAZ5 was found to promote flowering (Figures 5 and 6). Despite the inability to obtain stable genetically transformed materials for GhFL, the levels of MeJA were analyzed in SXY1 and its mutant dt2, revealing a significant increase in MeJA content in dt2 compared to the wild‐type SXY1 (P < 0.05, Student's t‐test) (Figure S9a). These findings suggest that the dt2 (GaFL) mutation may influence jasmonic acid metabolism or signaling. Analysis of the promoter regions of GhFL and GhSP2 identified the presence of MeJA‐responsive elements in the two genes, suggesting potential regulation by jasmonic acid (Figure S9d). And we hope to further analyze the function of both in response to jasmonate through subsequent studies.

The JA content was altered in GhSP2‐silenced plants, suggesting a potential regulatory role of GhSP2 in JA biosynthesis or signaling (Table S4). However, the precise mechanism by which GhSP2 participates in JA signaling remains unclear. To explore this further, we analyzed the promoter regions of both GhSP2 and GhJAZ5. Notably, we identified the presence of CArG box cis‐acting elements in both promoters (Figure S9e), which are known binding sites for MADS‐box proteins. Within the MADS‐box family, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) is a well‐characterized integrator of multiple flowering signals, including photoperiod, vernalization, phytohormone, and aging pathway (Srikanth & Schmid, 2011). While our data demonstrate that silencing GhSP2 elevates JA pathway metabolites and upregulates GhJAZ5, the precise molecular mechanism linking GhSP2 to JA homeostasis remains unclear. GhSP2, as a PEBP protein, is unlikely to directly regulate JA biosynthesis enzymes. One plausible hypothesis is that GhSP2 expression itself is modulated by upstream flowering integrators like GhSOC1, potentially placing GhSP2 within a regulatory cascade that indirectly influences JA synthesis or signaling.

Although this study primarily focuses on the jasmonic acid (JA) signaling pathway, the complex plant architecture development in cotton involves synergistic interactions with multiple phytohormones, including gibberellin (Zhong et al., 2024) and abscisic acid (Zhan et al., 2021). Future studies should employ integrated hormone profiling to elucidate whether GhFL‐GhSP2 precisely coordinates branching architecture and flowering time by integrating JA signaling with other hormonal pathways. Additionally, the functional divergence of homologous genes from the A and D subgenomes in cotton and their potential influence on JA pathway responsiveness warrant further investigation.

In summary, the dt2 mutant of G. arboreum is caused by a single base mutation in the GaFL gene, while GhFL, the homolog of GaFL in G. hirsutum, shares similar functions. Silencing GhFL in G. hirsutum resulted in a terminal flower phenotype at the shoot apex. Besides, GhFL interacted with GhSP2, which was demonstrated to be a negative regulator of the flowering process. The JA content and the expression of GhJAZ5 were upregulated in GhSP2‐silenced plants, implying a potential association between JA signaling and flowering regulation. These results elucidate GhSP2 as a key regulator of flowering time and demonstrate a physical interaction between GhFL and GhSP2. This interaction suggests the existence of a regulatory module integrating signals to coordinate both plant architecture and flowering time in cotton (Figure 7).

Figure 7.

Figure 7

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The hypothesis model of functional mechanism of GhFL and GhSP2 in plant architecture regulation.

Silencing GhFL in G. hirsutum led to the emergence of terminal flowers on monopodial shoot apical meristems (SAMs). In contrast, silencing GhSP2 resulted in an earlier flowering time. Furthermore, a decrease in GhSP2 expression was directly correlated with lowered jasmonic acid (JA) levels and an elevated transcription rate of GhJAZ5, suggesting a possible interaction between JA signaling pathways and flowering regulation. The diagram accompanying these insights employed blue triangles to signify both monopodial and sympodial SAMs, black arrows to represent vegetative branches, and red circles to denote bolls.

MATERIALS AND METHODS

Plant materials

The main experimental materials used in this research were: G. hirsutum acc. TM‐1, ZM425 and G. arboreum acc. SXY1. TM‐1 with normal branches and an indeterminate growth habit. ZM425 is an early maturing variety. SXY1 is an indeterminate growth plant, with indeterminate inflorescence and branches, which was used as a background material to obtained determinate growth mutant dt2 by EMS mutagenesis. G. arboreum acc. DQJ is an indeterminate growth plant which was used as paternal material to construct a F2 population. All the plants were kept in the National medium‐term Gene Bank of Cotton in China and were planted in the field of the Institute of Cotton Research of Chinese Academic Agricultural Sciences (ICR, CAAS), Anyang, Henan, China. G. hirsutum YZ1, OE‐JAZ and RNAi‐JAZ were supplied by the College of Plant Science and Technology at Huazhong Agricultural University, Wuhan, Hubei, China. All plant materials in this research were planted in the greenhouse under a 16/8‐h light/dark cycle (28–35°C during the day and 20–25°C at night).

Identification of the determinate growth 2 in EMS‐induced mutation population of G. arboreum

To generate a diverse range of mutants with novel traits, approximately 15 000 SXY1 seeds were pre‐soaked in phosphate buffer for 12 h at 28°C. Subsequently, the seeds underwent soaking in a 0.6% ethyl methane sulfonate (EMS) phosphate buffer solution for 8 h in darkness at 28°C, with continuous agitation throughout the duration. Ultimately, they were planted in the experimental field of ICR, CAAS (Sanya). Following four generations of self‐fertilization, 123 individual lines exhibiting distinct phenotypes were identified from 5980 M7 plants, one of which displayed determinate growth and was hence named determinate growth 2 (dt2).

F2 population construction and super bulked segregant analysis (BSA) sequencing

To clone the causal locus, the dt2 and DQJ were crossed to construct a hybrid line (200 F1 lines) in 2020, and an F2 population (1803 plants) was generated by self‐fertilization of the F1 population in 2021. Fresh young leaves were collected from parental lines (DQJ and dt2), 50 F2 individual plants exhibiting indeterminate growth and 50 F2 individual plants exhibiting determinate growth. Genomic DNA was extracted from the leaves following the previously reported method (Gong et al., 2018; Li et al., 2019). Two DNA pools were constructed. One pool consisted of DNAs from 50 F2 plants showing the dominant phenotype (indeterminate growth), and another pool consisted of DNAs from 50 F2 plants showing the recessive phenotype (determinate growth). The experimental process is performed according to the standard protocol provided by Illumina for BSA‐sequencing. The DNA of each sample was randomly broken into 350 bp fragments by ultrasonic fragmentation to construct the sequencing library. The library was sequenced by Illumina HiSeq after passing the quality inspection. Each sample was sequenced at 30× coverage of the assembled genome with 150‐bp paired‐end reads. Clean reads were mapped to the G._arboreum (A2) ‘SXY1’ genome CRI‐updated_v1 (https://www.cottongen.org/species/Gossypium_arboreum/CRI‐A2_genome_v1.0) by BWA after filtering the raw reads. According to the positioning results of clean reads in the reference genome, Picard was used to mark duplicates, GATK for local realignment, base recalibration and other preprocessing to ensure the accuracy of the detected SNPs. The single nucleotide polymorphism was further detected by GATK and filtered to obtain the final SNP site set. BSA‐sequencing analysis was performed using the runQTLseqAnalysis method of QTLseqr for SNP ratio calculation, and the average ΔSNP index was calculated based on a 1000 kb sliding window with a 10 kb step size to identify regions associated with the target trait.

Development and utilization of KASP marker in the GaDT2 cloning

According to the BSA result, we found a significant single signal on Chr07 indicating that the determinate growth trait was controlled by a single locus, and designed KASP markers within the candidate region (Chr07: 3.79 Mb to 6.60 Mb). The KASP marker development was performed by following a previous workflow (Liu et al., 2023).

Identification of the FL genes in G. hirsutum

The G. hirsutum (version ICR_v1.0) reference genome, protein‐coding sequences, and functional annotations were retrieved from the CottonGen Database (Yu et al., 2014). For the LEAFY (LFY) transcription factor family, two conserved domains HMM profiles (PF01698 and PF17538) were downloaded from the Pfam database (http://pfam‐legacy.xfam.org/). We performed HMM‐based screening using TBtools (version 2.0) with a stringent E‐value cutoff (<1e‐5), revealing evolutionarily conserved LFY‐domain genes whose genomic architectures and chromosomal locations were graphically mapped (Chen et al., 2023).

Analysis of cis‐acting elements in promoters

The 2000 bp promoter sequences upstream of the ATG start the CDS of GhFL, GhSP2, and GhJAZ5 were extracted from the G. hirsutum (version ICR_v1.0) genomic sequence files. The extracted promoter sequences were analyzed for cis‐acting elements using the PLANTCARE online tool (Lescot et al., 2002). The identified cis‐acting elements were visualized using TBtools.

qRT‐PCR analysis

RNA extraction from various tissues was conducted using the FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). Total RNA was converted to cDNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio Inc., Beijing, China). qRT‐PCR assays were carried out with the 2 × RealStar Green Fast Mixture with ROX II (GenStar, Beijing, China) in a volume of 20 μl. The PCR reaction protocol was as follows: Initial denaturation at 95°C for 2 min, followed by 40 cycles of amplification at 95°C for 15 sec and 60°C for 30 sec. All primer sequences used for gene expression analysis are listed in Table S1. The qRT‐PCR analysis was conducted with three biological replicates, followed by the application of the 2−ΔΔCt method to quantify the relative gene expression levels (Livak & Schmittgen, 2001).

Virus‐induced gene silencing (VIGS) assay

Primers targeting the CDS of candidate genes were designed and 300–400 bp specific fragment were amplified. Gene specific fragments were inserted into the pTRV2 vector and confirmed by Sanger sequencing. The vectors pTRV2‐GaFL, pTRV2‐GhFL, pTRV2‐GhSP2, and pTRV1 were transformed into Agrobacterium tumefaciens (A. tumefaciens) strain GV3101. The transformed A. tumefaciens were incubated overnight at 28°C in an antibiotic selective medium containing 50 mg/ml each of rifampicin, gentamicin, and kanamycin. Following incubation, the cells were harvested and resuspended in a solution containing 10 mm MgCl2, 10 mm MES, and 200 μm acetosyringone. The optical density at 600 nm (OD600) of the suspension was adjusted to 1.0. The cotton cotyledons were then injected once they were fully expanded (Gao et al., 2011).

Plasmids construction and transformation of Arabidopsis

The full‐length CDS of GhFL and GhSP2 was cloned into the plasmid pRI101 and subsequently introduced into A. tumefaciens GV3101 cells. Arabidopsis thaliana was transformed via floral dip method. Transgenic plants were selected on Murashige and Skoog (MS) medium supplemented with 50 mg/ml kanamycin and verified by polymerase chain reaction (PCR). Kanamycin‐resistant plants were transplanted, with non‐transgenic wild‐type (WT) seedlings used as controls for phenotypic analysis.

Subcellular localization assay

The CDS sequence of GhFL (excluding the stop codon) was cloned into the pRI101 plasmid to construct the 35S: GhFL‐GFP fusion vector. The recombinant vector and the control vector were subsequently transformed into A. tumefaciens GV3101 cells. Then, A. tumefaciens was injected into the abaxial surface of N. benthamiana leaves. The completed injected N. benthamiana was incubated in a dark environment for 24 h and then transferred to a normal photoperiod (16 h light/8 h dark) for 2 days. One square centimeter of N. benthamiana leaf tissue was taken by adding a drop of sterile water and then covering the tissue with a coverslip, followed by detection of GFP fluorescence by confocal microscopy.

Bimolecular fluorescence complementation (BiFC) assay

The CDS of GhFL was subcloned into the p326‐106 vector, while GhSP2 was inserted into the p326‐104 vector. These constructs were subsequently introduced into cotton protoplasts. The method of protoplasts isolation and transformation from TM‐1 cotyledon was slightly modified as described previously (Sun et al., 2024). Briefly, the chlorotic cotyledon was cut into 0.5–1 mm strips and dipped into digestion solution (1.5% Cellulase R10, 0.4% Macerozyme R10, 1% Hemi Cellulase, 0.4 M Mannitol, 20 mm KCl, 20 mm MES [pH 5.7], 10 mm CaCl2, and 0.1% BSA). The vectors (6 μg) were transformed in protoplasts using PEG 4000 (Sigma‐Aldrich Shanghai Trading Co., Ltd, Shanghai, China). The transformed protoplasts were cultured at 28°C in the dark for 16 h.

GST pull‐down

GhFL was cloned into pGEX‐4T vector as baits. The construct was transformed into the Escherichia coli BL21 (DE3) strain to produce recombinant proteins. GhSP2 was cloned into pRI101 vector containing the GFP protein tag as preys. The construct was transformed into A. tumefaciens GV3101 cells and expressed in N. benthamiana leaves. The pGEX‐4T vector was employed to express glutathione‐S‐transferase (GST) as a negative control. Bait and prey proteins were incubated at 4°C for 6 h and subsequently purified using glutathione‐conjugated agarose beads. The mixed proteins were then separated on 12% SDS‐PAGE and followed by immunoblotting analysis using anti‐GST antibody.

Luciferase (LCI) complementation assay in N. benthamiana

For bimolecular luciferase complementation assays, CDS of GhFL and GhSP2, were cloned into pCAMBIA1300 plasmids to generate the 35S: GhFl‐cLuc, 35S: GhSP2‐nLuc and 35S: GhSP2‐nLuc fusion vector, respectively. A. tumefaciens (GV3101) bacteria containing the indicated constructs were transiently transformed into the leaves of N. benthamiana. The LUC image is captured using the Tanon 5200 Plant Imaging System.

Measurement of phytohormones contents

Phytohormone levels were analyzed by MetWare (http://www.metware.cn/) utilizing the AB Sciex QTRAP 6500 LC–MS/MS platform. Fresh plant samples were harvested, immediately flash‐frozen in liquid nitrogen, pulverized into powder (30 Hz, 1 min), and stored at −80°C until further use. A 50 mg aliquot of the plant sample was placed into a 2 ml plastic microtube, frozen in liquid nitrogen, and dissolved in a methanol/water/formic acid solution (15:4:1, V/V/V). An internal standard mixture (10 μl, 100 ng/ml) was added to the extract for quantification purposes. The mixture was vortexed for 10 min, followed by centrifugation for 5 min at 12000 rpm and 4°C. The supernatant was transferred to clean plastic microtubes, evaporated to dryness, and subsequently dissolved in 100 μl of 80% methanol (V/V). It was then filtered through a 0.22 μm membrane filter for further LC–MS/MS analysis. The sample extracts were analyzed using a UPLC‐ESI‐MS/MS system (UPLC: ExionLC™ AD from https://sciex.com.cn/; MS: Applied Biosystems 6500 Triple Quadrupole from https://sciex.com.cn/). The analytical conditions were as follows: LC column: Waters ACQUITY UPLC HSS T3 C18 (100 mm × 2.1 mm i.d., 1.8 μm); solvent system: water with 0.04% acetic acid (A) and acetonitrile with 0.04% acetic acid (B); gradient program: starting at 5% B (0–1 min), increasing to 95% B (1–8 min), maintaining at 95% B (8–9 min), and finally returning to 5% B (9.1–12 min); flow rate: 0.35 ml/min; temperature: 40°C; injection volume: 2 μl.

AUTHOR CONTRIBUTIONS

ZG: Conceptualization, validation, data curation, writing—original draft. QC: Validation, data curation, writing—original draft. PM: Validation, data curation, writing—original draft. HC: Validation. QW: Validation. JL: Validation. GS: Conceptualization, writing—review and editing, funding acquisition. DZ: Validation.

CONFLICT OF INTEREST

The authors declare no competing interests.

DISCLOSURE

The authors acknowledge the use of ChatGPT‐4 (accessed January 2025), DeepSeek v1.0 (accessed April and July 2025), DeepSeek v2.0.5 (accessed September 2025), to polish language, correct grammar and spelling, suggest word choices, and rephrase awkward sentences. All AI‐generated language polishing suggestions were reviewed, revised, and approved by the authors, who take full responsibility for the accuracy and integrity of the work.

Supporting information

Figure S1. The plant height of SXY1 and dt2 plants. The error bars indicate mean ± SEM of three independent experiments. **P < 0.01, (Student's t‐test).

Figure S2. Integrative Genomics Viewer (IGV) analysis for the resequencing data of the 8 candidate genes. The SNP sites between the parent and the reference genome were marked by black dotted box.

Figure S3. A 452 bp sequence alignment of Ga07G0556 between the dt2 mutant and wild type Shixiya 1. A SNP (G to A) was observed in the dt2 mutant.

Figure S4. GhFL‐At (GhFL) is the homolog of GaFL in G. hirsutum.

Figure S5. Phenotypic differences between TRV: GhFL and TRV: GhSP (Scale bar = 15 cm).

Figure S6. GhSP2 heterologous overexpression in Arabidopsis.

Figure S7. GhSP2 controls the JA content and alters the expression levels of and JAZ in cotton.

Figure S8. Amino acid sequence alignment and phylogenetic analysis of two GhSPs and CEN, TFL1.

Figure S9. Analysis of MeJA content, GaSP2 expression patterns, and cis‐acting elements of GhFL, GhSP2 and GhJAZ5 promoters.

TPJ-127-0-s002.doc (50.1MB, doc)

Table S1. The primer in this study.

Table S2. Chi‐square test and genetic analysis of F2 population.

Table S3. The list of the candidate genes.

Table S4. Alterations in hormone content of TRV: GhSP2 plants.

TPJ-127-0-s001.doc (149.5KB, doc)

ACKNOWLEDGMENTS

This work was supported by the National Key R&D Program of China (2024YFD1200305) and China Agriculture Research System (Grant No. CARS‐15‐06) and Natural Science Foundation of Henan Province (Grant No. 232300421041 and 252300423099) and Nanfan Special Project (Grant No. YBXM2546, YBXM2564 and YBXM2634) and State Key Laboratory of Cotton Bio‐breeding and Integrated Utilization/ Institute of Cotton Research of CAAS (Grant No: CB2025C18) and ICR‐CAAS Special Fund for Basic Research (Grant No: 1610162026032). The funders had no role in the design of the study, collection, analysis, or interpretation of the data, the writing of the manuscript, or the decision to submit the manuscript for publication. We are grateful to Huazhong Agricultural University for providing us with the cotton accessions OE‐JAZ, YZ1, and RNAi‐JAZ.

Contributor Information

Ji Liu, Email: liuji@caas.cn.

Guoli Song, Email: sglzms@163.com.

Dongyun Zuo, Email: zdy041@163.com.

DATA AVAILABILITY STATEMENT

All relevant data required to replicate the findings of this study are included within this manuscript and its supplementary materials. For further inquiries or requests for additional materials, please contact the corresponding author at zdy041@163.com.

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Associated Data

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

Supplementary Materials

Figure S1. The plant height of SXY1 and dt2 plants. The error bars indicate mean ± SEM of three independent experiments. **P < 0.01, (Student's t‐test).

Figure S2. Integrative Genomics Viewer (IGV) analysis for the resequencing data of the 8 candidate genes. The SNP sites between the parent and the reference genome were marked by black dotted box.

Figure S3. A 452 bp sequence alignment of Ga07G0556 between the dt2 mutant and wild type Shixiya 1. A SNP (G to A) was observed in the dt2 mutant.

Figure S4. GhFL‐At (GhFL) is the homolog of GaFL in G. hirsutum.

Figure S5. Phenotypic differences between TRV: GhFL and TRV: GhSP (Scale bar = 15 cm).

Figure S6. GhSP2 heterologous overexpression in Arabidopsis.

Figure S7. GhSP2 controls the JA content and alters the expression levels of and JAZ in cotton.

Figure S8. Amino acid sequence alignment and phylogenetic analysis of two GhSPs and CEN, TFL1.

Figure S9. Analysis of MeJA content, GaSP2 expression patterns, and cis‐acting elements of GhFL, GhSP2 and GhJAZ5 promoters.

TPJ-127-0-s002.doc (50.1MB, doc)

Table S1. The primer in this study.

Table S2. Chi‐square test and genetic analysis of F2 population.

Table S3. The list of the candidate genes.

Table S4. Alterations in hormone content of TRV: GhSP2 plants.

TPJ-127-0-s001.doc (149.5KB, doc)

Data Availability Statement

All relevant data required to replicate the findings of this study are included within this manuscript and its supplementary materials. For further inquiries or requests for additional materials, please contact the corresponding author at zdy041@163.com.

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