Integrative transcriptomic and metabolomic analyses reveal mechanisms underlying calcium-induced enhancement of female flower production in Cucumis sativus L.

Abstract

Cucumber sex differentiation is a complex process regulated by multiple factors, including calcium (Ca²⁺) signaling. Although Ca²⁺ has been implicated in flower sex determination, its precise regulatory mechanisms remain unclear. In this study, the androecious cucumber line Banna 31–10 was treated with CaCl₂ at the shoot apices at seven-day intervals for three applications (Ca-1W, Ca-2W, Ca-3W). CaCl₂ treatment significantly increased the number of female flowers, with the effect becoming more pronounced with prolonged application. Integrated transcriptomic and metabolomic analyses revealed extensive molecular and metabolic reprogramming in response to CaCl₂. Transcription factor (TF) analysis identified 347 TFs from 56 families, with ERF, bHLH, MYB, C2H2, and MADS-box families playing key roles. Integrated transcriptome–metabolome correlation highlighted the involvement of flavonoids and plant hormone pathways, including ethylene, abscisic acid, gibberellin, cytokinin, auxin, jasmonic acid, salicylic acid, and brassinosteroid. Notably, CsACS2 and multiple calmodulin-like (CML) genes were strongly associated with hormone biosynthesis and signaling, suggesting a central role of Ca²⁺-CML-mediated regulation in female flower differentiation. Weighted gene co-expression network analysis (WGCNA) identified hub genes in co-expression modules linked to CaCl₂ response, further supporting the regulatory network underlying sex differentiation. This study provides new insights into the role of the Ca²⁺ signaling system in cucumber sex differentiation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08338-8.

Highlights

*Female flower formation was enhanced following CaCl₂ treatment in cucumber

*Transcriptome and metabolome analyses reveal key DEGs, DAMs, and pathways

*ERF, bHLH, MYB, C2H2, and MADS-box TFs may be crucial regulators

*CML genes correlate with hormone biosynthesis and signaling

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08338-8.

Introduction

Cucumber (Cucumis sativus L.) is one of the most widely cultivated gourd vegetables worldwide. It is dominantly monoecious, with floral bud differentiation initiated as early as the second true leaf stage during the young seedling phase, which determines the developmental trajectory of male and female flower formation. Cucumber produces male, female, and bisexual flowers, with sex expression classified into seven types: monoecious, gynoecious, female, androecious, hermaphroditic, andromonoecious, and trimonoecious. Due to its diversity in floral sex types and sex expression, cucumber has become a classic model for studying plant sex differentiation [1]. Local cucumber varieties from Yunnan Province exhibit strong vegetative vigor and stress resistance but are predominantly androecious, which severely limits both fruit production and seed propagation. Since the formation and development of female flowers are essential for crop productivity, understanding the mechanisms underlying sex differentiation in Yunnan cucumber varieties is crucial for enhancing yield and seed production.

Plant sex differentiation is the outcome of complex interactions among genetic determinants, hormonal regulation, and environmental cues [2]. The inherent complexity of sex determination underlies the diversity of sex expression in plants. In cucumber, several genes, notably five, have been identified as key regulators of sex differentiation, including three ACS genes CsACS1G (F), CsACS2 (M) [3] and CsACS11 (A) [4]), one 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene (CsACO2), and one transfer factor of the Cys2-His2 (C2H2) zinc finger gene family (CsWIP1 (G)) [5, 6]. CsACS2 primarily suppresses stamen development and enables pistils to fully develop,CsACS11 stimulates the development of carpel; CsACO2 and CsACS11 promote ethylene production synergistically, and CsACO2 absence leads to the suppression of female flower development [7]. WIND1-Interacting Protein 1 (CsWIP1), as the inhibitor of carpel development, is inhibited by CsACS11, which can also inhibit the expression of CsACS2 and CsACO2 [4, 7, 8]. These genes regulate the development of distinct floral sexes by controlling the selective growth or cessation of reproductive organs. All the five major sex-determining genes in cucumber have been found to be associated with ethylene synthesis, confirming the central role of ethylene in cucumber sex determination [9].

As the final component of the ethylene signaling pathway, ethylene response factors (ERFs) play crucial roles in transcriptional regulation, underscoring their importance in plant developmental processes [10, 11]. In cucumber, CsERF31 has been reported to positively regulate the transcription of the M gene by binding to the ethylene-responsive element (ERE) in its promoter [12], thereby participating in ethylene-mediated sex determination. Furthermore, recent studies have observed the upregulation of CsERF23, CsERF78, and CsERF121 during female flower bud development. In contrast, CsERF69 and CsERF84 are responsive to cytokinins and other hormones, exhibiting increased expression during female flower bud differentiation and participating in ovary development. Beyond ethylene, other plant hormones also modulate sex expression. Gibberellins (GAs) play a significant role in cucumber sex determination [13], with exogenous GA application inducing the formation of male flowers in genetically female plants [14]. The GA signaling pathway has been implicated in stamen and anther development in hermaphroditic plants, as observed in Arabidopsis and rice (Oryza sativa) [15, 16, 17]. In contrast, abscisic acid (ABA) promotes femaleness by antagonizing GA activity [18]. Exogenous auxin also enhances femaleness in cucumber by activating sex-determining genes and promoting ethylene biosynthesis [19]. Furthermore, MCM1, AGAMOUS, DEFICIENS, and SRF-box (MADS-box) transcription factors (TFs), a conserved family characterized by a MADS DNA-binding domain, may influence sex differentiation by regulating stamen and ovary development in cucumber and melon [20–22]. Importantly, their expression is modulated by hormonal signals such as ethylene and GA, and they can, in turn, regulate hormone biosynthesis or signaling genes. This establishes a feedback loop that integrates hormonal and transcriptional control, coordinating multiple regulatory layers to determine flower sex in cucumber.

Calcium (Ca²⁺) is a multi-functional signaling ion that mediates plant responses to developmental cues, environmental stimuli, and hormonal signals [23, 24]. The perception and decoding of Ca²⁺ signals by Ca²⁺ sensor proteins are critical regulatory processes in the Ca²⁺ signaling pathway. Four major families of Ca²⁺ sensor proteins have been identified: calmodulins (CaMs), calmodulin-like proteins (CMLs), calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs) [25, 26]. Among these, the CML family has been reported to respond to diverse biotic and abiotic stimuli [25, 26]. They are also involved in key developmental processes, including pollen germination and pollen tube growth [26, 27], as well as vegetative growth, and hormone-mediated pathways [28].

Our previous yeast two-hybrid assays demonstrated that cucumber CsCML8 interacts with the proteins encoded by the key female flower gene CsACS1G (F) and the key male flower gene CsACS2 (M), suggesting that CMLs may be involved in sex differentiation in cucumber. Additionally, we observed an association between cucumber CMLs and C2H2 signal transduction. CML25-like was found to be able to stimulate parthenocarpy and promote fruit enlargement, likely by upregulating hormone-related genes in the ovaries of parthenocarpic cucumber [29].

At present, reports on how the Ca²⁺ signaling system regulates sex differentiation in cucumber remain limited. To address this gap, we aimed to elucidate the regulatory network controlling sex differentiation and investigate the involvement of Ca²⁺ signaling in this process. Using the androecious cucumber line ‘Banna 31–10’, we applied CaCl₂ treatments to examine changes in female flower numbers and associated molecular alterations. Our study offers a better understanding of the cucumber sex differentiation network and a theoretical foundation for breeding strategies.

Material and method

Plant material and CaCl_₂ treatment

The androecious cucumber line ‘Banna 31–10’ was used as experimental material. This line belongs to Xishuangbanna cucumber (Cucumis sativus var. xishuangbannanesis), a unique Chinese cucumber variant characterized by its orange-yellow mature fruit flesh. It has long been cultivated and utilized by local ethnic groups in Yunnan Province.

Cucumber seedlings were cultivated in a growth chamber under controlled conditions (25/20 °C, day/night; 12/12 h photoperiod; 70% relative humidity). Our preliminary experiments with a range of CaCl₂·2H₂O concentrations showed that 10 mM was optimal for inducing female flower formation (Table S1). At the two-leaf stage, seedlings received a foliar application of 10 mM CaCl₂·2H₂O (GHTECH, Shantou, Guangdong, China) to full coverage using a hand-held sprayer. The first sampling was conducted one week after this initial treatment, and three seedlings were collected (Ca-1W). On the same day of first sampling, the remaining seedlings received the second CaCl₂ treatment (April 7, 2024). After one week, the second sampling was carried out (Ca-2W), immediately followed by the third CaCl₂ treatment (April 14, 2024). Seedlings that received all three treatments were sampled one week later on April 21, 2024 (Ca-3W). Therefore, Ca-1W corresponded to two-leaf-stage seedlings treated once; Ca-2W corresponded to three-leaf-stage seedlings treated twice; and Ca-3W corresponded to four-leaf-stage seedlings treated three times. Distilled water was applied on the same schedule for the control group. At each sampling point, shoot apical meristems (SAMs) were collected as three biological replicates, immediately frozen in liquid nitrogen, and stored at − 80 °C for further analysis.

The plant was identified by Professor Junqiang Xu (College of Landscape and Horticulture, Yunnan Agricultural University), and voucher specimens were deposited in the laboratory of Yunnan Agricultural University. All experimental research involving plants, including the collection of plant material, was conducted in accordance with institutional, national, and international guidelines. Field studies were carried out following local legislation, and all necessary permissions and/or licenses for the collection and use of plant material were obtained from the relevant authorities.

RNA sequencing (RNA-seq) and data analysis

Total RNA was extracted from cucumber SAMs using the ethanol precipitation method and the CTAB-PBIOZOL protocol. RNA integrity and potential DNA contamination were assessed via agarose gel electrophoresis. RNA purity was determined using a NanoPhotometer by measuring OD260/280 and OD260/230. RNA concentration was precisely measured using a Qubit 4.0 fluorometer/MD microplate reader. RNA integrity was further analyzed using a Qsep400 bioanalyzer.

Sequencing libraries were prepared using a standard Illumina-stranded protocol, including size selection for insert fragments of 250–350 bp. Library quality was verified by assessing fragment size distribution (Qsep400 bioanalyzer) and accurately quantified via quantitative polymerase chain reaction (q-PCR). Libraries meeting quality thresholds were sequenced on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) using a paired-end 150 bp (PE150) strategy, with a sequencing depth of approximately 6 Gb of clean data per sample. Low-quality sequences were removed from raw data using Fastp [30]. Ultimately, reads with Qphred ≤ 20 were discarded, accounting for over 50% of the total read length. Clean reads were then aligned to the reference genome using HISAT2 [31]. Gene alignment was quantified using featureCounts [32], and gene expression levels were quantified as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Differential expression analysis was performed using DESeq2 [33, 34]. The DESeq method was applied for normalization, and adjusted p-values were calculated using the Benjamini–Hochberg (BH) procedure to control the false discovery rate. Differentially expressed genes (DEGs) were identified with adjusted P ≤ 0.05 and |log2 fold change|≥ 1. Functional annotation of genes was obtained using DIAMOND [35] against the RefSeq non-redundant proteins (NR) database, the Swiss-Prot database, the Gene Ontology (GO) database, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, with an E-value < 1e-5. TF annotation was conducted using iTAK [36] (E-value < 1e-5).

Metabolite extraction and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis

Freeze-dried samples were ground to a fine powder at 30 Hz for 1.5 min using a grinder (MM 400; Retsch Haan, Germany). Subsequently, 50 mg of the powder was mixed with 1200 μL of a pre-cooled (−20 °C) 70% methanolic aqueous solution containing internal standard. The mixture was vortexed for 30 s at 30-min intervals, repeated six times. After centrifugation at 12,000 rpm for 3 min, the supernatant was carefully collected, filtered through a microporous membrane (0.22 μm of pore size), and transferred to an injection vial for subsequent analysis using a UPLC-ESI–MS/MS system (ExionLC™ AD, SCIEX, Framingham, MA, USA). Chromatographic separation was performed using an Agilent SB-C18 column (100 mm × 2.1 mm, 1.8 µm) (Agilent Technologies, Santa Clara, CA, USA). The mobile phase consisted of solvent A (pure water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The gradient elution program started with 95% A and 5% B, then linearly increased to 95% B over 9.0 min, held for 1.0 min, and returned to the initial composition (95% A and 5% B) within 1.1 min and held for an additional 2.9 min. The flow rate was 0.35 mL/min, and the injection volume was 2 μL. The effluent was alternatively directed to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS. The ESI source was operated under the following parameters: source temperature of 550 °C; ion spray voltage (IS) set at 5500 V (positive ion mode)/−4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) set at 50, 60, and 25 psi, respectively. The collision-activated dissociation (CAD) was set to high. Declustering potential (DP) and collision energy (CE) were optimized for each MRM transition, and a specific set of transitions was monitored according to the elution time of corresponding metabolites.

Metabolites were identified using a self-built MetWare Database (MWDB) based on secondary mass spectra. Isotopic signals, repeated ions, and fragment duplicates were removed. For metabolite quantification, a triple quadrupole mass spectrometry (QQQ) system operating in multiple reaction monitoring (MRM) mode was employed to enhance specificity, with nitrogen as the collision gas set to medium. Raw data were processed using the MultiQuant software (Sciex, Framingham, MA, USA), which performed chromatographic peak area integration and inter-sample calibration (retention time alignment and peak shape optimization) to standardize the quantification criteria for the same metabolite across different samples. The integrated peak area of each metabolite was used to quantify its relative content in the sample. Missing values were imputed with one-fifth of the minimum value per metabolite. Only metabolites with CV < 0.5 in quality control (QC) samples were retained. Data were unit variance (UV)-scaled for PCA and clustering analyses, and mean-centered and log₂-transformed prior to OPLS-DA to improve normality. Quality control was confirmed via TIC overlap of QC samples and CV distribution, ensuring reproducibility for downstream analyses.

Orthogonal partial least squares discriminant analysis (OPLS-DA) was performed using the MetaboAnalystR package [37]. To ensure model reliability, a permutation test with 200 iterations was performed to avoid overfitting. Differentially accumulated metabolites (DAMs) were identified with variable importance in projection (VIP) > 1 and fold change ≥ 2 or ≤ 0.5. These DAMs were subsequently annotated using the KEGG Compound Database (http://www.kegg.jp/kegg/compound/). The annotated metabolites were mapped to the KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html). Significantly enriched metabolic pathways were identified using metabolite set enrichment analysis (MSEA), with statistical significance determined by p-values derived from hypergeometric test.

Correlation analysis of transcriptome and metabolome data

Pairwise Pearson correlations were calculated across all samples for DEG–DAM and DEG–DEG pairs using the cor function in R. Significant correlations were defined as |r|> 0.85 with adjusted P < 0.05. Significant pairs were imported into Cytoscape for network visualization. Hub genes were identified based on degree values, representing the number of significant connections, with higher-degree nodes considered as hubs. Correlated pairs were mapped to the KEGG database to highlight pathways potentially co-regulated at transcriptomic and metabolomic levels.

WGCNA

Lowly expressed and non-variable genes were filtered using the varFilter function in the genefilter package in R. A weighted co-expression network was constructed using WGCNA based on the gene expression matrix. The optimal soft-thresholding power was determined using the pickSoftThreshold function in the WGCNA package in R (RsquaredCut = 0.85). The eigengene of each module was calculated and used to assess its association with each sample. Co-expression networks were visualized in Cytoscape (v. 2.8).

Detection of phytohormones

Samples were ground into fine powder using a tissue grinder (30 Hz, 1 min). Subsequently, 50 mg of the homogenized material was transferred into a centrifuge tube and mixed with 10 μL of internal standard solution (100 ng/mL; purity > 99%, OlChemIm, Czechoslovakia) and 1 mL of extraction solvent (methanol/water/formic acid, 15:4:1, v/v/v). The mixture was vortexed for 10 min and centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was collected, diluted with 100 μL of 80% methanol, filtered through a 0.22 μm membrane, and analyzed using an ExionLC™ AD UPLC system coupled to a QTRAP® 6500 + triple quadrupole mass spectrometer (SCIEX, Framingham, MA, USA). Chromatographic separation was achieved on a Waters ACQUITY UPLC HSS T3 C18 column (100 × 2.1 mm, 1.8 μm; Waters, Milford, MA, USA). The mobile phases consisted of (A) water with 0.04% acetic acid and (B) acetonitrile with 0.04% acetic acid. The gradient elution program was as follows: 0–1.0 min, 95% A; 1.0–8.0 min, linear decrease to 5% A; 8.0–9.0 min, 5% A; 9.0–9.1 min, return to 95% A; and 9.1–12.0 min, re-equilibration at 95% A. The flow rate was 0.35 mL/min, the column temperature was set at 40 °C, and the injection volume was 2 μL.

The mass spectrometer was operated with an electrospray ionization (ESI) source at 550 °C. Curtain gas was set at 35 psi. Metabolite identification was based on the Metware Database (MWDB), constructed from authenticated reference standards. Quantification was performed using MRM peak integration, and concentrations were calculated from calibration curves generated from corresponding standard solutions.

Quantitative reverse transcription PCR (qRT-PCR)

Total RNA was extracted from cucumber SAMs using the Tiangen RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized from up to 2 µg RNA using the All-In-One 5X RT MasterMix with gDNA Removal (abm, Richmond, Canada). qRT-PCR was carried out using the EvaGreen 2X qPCR MasterMix (abm, Richmond, Canada) on a Bio-Rad CFX9 Real-Time System (Bio-Rad, Hercules, CA, USA). Each 20 µL reaction contained 10 µL MasterMix, 0.6 µL each of forward and reverse primers (10 µM; final concentration 300 nM), cDNA template, and nuclease-free water. Primers were designed using Primer 5.0 (Table S2). The thermal cycling conditions were: 95 °C for 10 min, followed by 30 cycles of 95 °C for 15 s and 60 °C for 60 s. Each gene was analyzed in triplicate, and relative expression levels were calculated using the 2^ − ΔΔCt method.

Results

CaCl₂ treatment increases the number of female flowers in cucumber

Cucumber seedlings of the androecious line Banna 31–10 were treated with CaCl₂ to investigate its effect on female flower development (Fig. 1A). The number of female flowers within the first 25 nodes of each plant was recorded. Statistical analysis showed that all CaCl₂ treatment groups (Ca-1W, Ca-2W, and Ca-3W) exhibited a significant increase in female flower numbers compared with the control group. Moreover, the number of female flowers increased with the duration of CaCl₂ treatment (Fig. 1B), indicating that prolonged CaCl₂ application promotes female flower differentiation in cucumber.

Fig. 1.

CaCl₂ treatment promotes female flower development in cucumber. A Cucumber seedlings of the androecious line Banna 31–10 were treated with 10 mM CaCl₂·2H₂O. Ca-1W: two-leaf-stage seedlings treated once; Ca-2W: three-leaf-stage seedlings treated twice; Ca-3W: four-leaf-stage seedlings treated three times. B CaCl₂ treatment increases the number of female flowers in cucumber. Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001 (Student’s t-test)

CaCl₂ treatment leads to transcriptomic alterations in cucumber

To elucidate the molecular basis of Ca²⁺-regulated sex differentiation in cucumber, we performed transcriptome sequencing using RNA-seq on cucumber seedlings treated with CaCl₂ solution. A total of 656,007,744 clean reads were obtained from 12 samples, with each sample generating ≥ 6 Gb of data, yielding 98.4 Gb of clean bases. The sequencing quality was high, with Q20 and Q30 scores ≥ 97.81% and 93.36%, respectively. Most clean reads were successfully mapped to the cucumber reference genome, including 94.31–97.71% uniquely mapped reads and 3.37 − 4.00% multiple-mapped reads (Table S3). These results indicate the high quality of the sequencing data, supporting subsequent bioinformatics analyses.

A total of 22,438 transcripts were detected across the 12 samples. Principal component analysis (PCA) was conducted to obtain an overview of the transcriptomic landscape. PC1 and PC2 explained 29.79% and 22.26% of the total variance, respectively. The PCA plot revealed a clear separation among groups, with biological replicates clustering together, indicating high sampling reliability and experimental consistency (Fig. 2A). Based on |log₂ fold change|≥ 1 and FDR < 0.05, a total of 2,987 DEGs were identified across all comparisons. The numbers of DEGs in Ca-1W and Ca-2W compared with CK were comparable, while Ca-3W showed a pronounced increase. (Fig. 2B). Heatmap analysis of both the global transcriptome profiles (Fig. S1A) and the top 50 commonly expressed genes (Fig. 2C) revealed distinct expression patterns among groups. Among the DEGs identified in the three treatment groups, 451 were shared. KEGG pathway enrichment analysis of these overlapping DEGs showed significant enrichment in secondary metabolism and plant signal transduction pathways (Fig. 2D).

Fig. 2.

Global transcriptomic responses of cucumber Banna 31–10 after CaCl₂ treatment. A Principal component analysis (PCA) of transcriptome data. B Numbers of upregulated and downregulated differentially expressed genes (DEGs) in different comparison groups (adjusted P ≤ 0.05 and |log2 fold change|≥ 1). C Heatmap analysis of the top 50 commonly expressed genes across groups. The color scale represents relative expression: yellow indicates higher expression and green indicates lower expression. D Venn diagram showing the numbers of DEGs unique to and shared among the comparison groups, along with KEGG enrichment analysis of the shared DEGs. E K-means clustering of DEGs illustrating six distinct expression patterns, with the number of genes in each pattern indicated

K-means clustering analysis grouped the DEGs into six distinct clusters, designated as T1–T6. T1 decreased from CK to Ca-1W, then stabilized and slightly recovered. T2 remained stable initially but subsequently declined continuously in the treatment groups. T3 peaked in the Ca-1W group, dropped in Ca-2W, and rebounded in Ca-3W. T4 decreased in Ca-1W, reached its maximum in Ca-2W, and slightly fell in Ca-3W. Both T5 and T6 exhibited an upward trend, with T6 remaining stable until Ca-2W before increasing sharply at Ca-3W (Fig. 2E).

CaCl₂ treatment leads to metabolic alterations in cucumber

To investigate metabolic changes induced by CaCl₂ treatment in cucumber, UPLC-MS/MS was used to profile metabolites of all samples. A total of 1,363 metabolites were detected. Consistent with transcriptome findings, PCA showed significant differences among groups, with PC1 and PC2 explaining 43.22% and 13.4% of the variance, respectively (Fig. 3A). DAMs were screened based on VIP > 1 and fold change ≥ 2 or ≤ 0.5, resulting in the identification of 668 DAMs. These included 230 flavonoids, 95 phenolic acids, 68 esters, 48 lignans and coumarins, 40 terpenoids, and 28 amino acids and derivatives (Fig. 3B). Heatmap analysis of both the global metabolite profiles (Fig. S1B) and the top 50 common metabolites (Fig. 3C) showed minimal differences between Ca-1W and CK, whereas Ca-2W and Ca-3W exhibited contrasting accumulation patterns relative to CK. Spefically, 227 DAMs were identified in Ca-1W, with 183 upregulated and 44 downregulated. Specifically, syringic acid-4-O-(6''-feruloyl)glucoside and 3'-O-Feruloyl swertiamarin were upregulated, whereas uralenol and 3-galloylquinic acid were downregulated. In Ca-2W, metabolites such as syringicacid-4-O-(6''-feruloyl)glucoside and glucosyl-5-O-glucoside were upregulated, whereas uralenol and phylloquinone (vitamin K1) were downregulated. In Ca-3W, 513 DAMs were identified, comprising 368 upregulated and 145 downregulated. Notably, hydroxy ricinoleic acid, and glucosyl-5-O-glucoside were upregulated, whereas sophorotrioside was downregulated (Fig. 3D).

Fig. 3.

Metabolome analysis of cucumber Banna 31–10 following CaCl₂ treatment. A. PCA of metabolomic data showing separation among treatment groups. B Classification of detected metabolites. C Heatmap analysis of the top 50 common metabolites across groups. The color scale represents relative expression: yellow indicates higher expression and green indicates lower expression. D Volcano plot of differentially accumulated metabolites (DAMs) in Ca-1W, Ca-2W, and Ca-3W compared with CK (VIP > 1 and fold change ≥ 2 or ≤ 0.5). E K-means clustering of DAMs identified six distinct accumulation patterns, with the number of metabolites noted in each cluster

K-means clustering analysis grouped the identified metabolites into six distinct clusters (M1–M6) based on their accumulation patterns. Metabolites in M1 gradually decreased in response to CaCl₂ treatment, with the highest levels observed in CK. M2 comprised metabolites that were slightly upregulated in Ca-1W but remained stable in subsequent treatments. M3 included metabolites showing marked increases in Ca-2W, followed by a sharp decrease in Ca-3W. In contrast, M4 contained metabolites that were progressively upregulated from Ca-2W to Ca-3W, while M5 exhibited a gradual increase as the number of CaCl₂ treatments increased. M6 encompassed metabolites that were initially downregulated in Ca-1W but subsequently increased with prolonged CaCl₂ treatment. Overall, these results indicate that metabolite accumulation patterns varied depending on the number of CaCl₂ applications, with clusters M4–M6 showing the most pronounced upregulation (Fig. 3E).

Predicted TF families involved in Ca²⁺-mediated regulation of cucumber sex differentiation

To investigate potential regulatory mechanisms, TFs were predicted with the DEGs using iTAK software. A total of 347 TFs were identified, belonging to 56 distinct TF families. The family with the largest number of DEGs was ERF, comprising 38 members, followed by bHLH (32 members) and MYB (23 members), while the remaining families contained between 1 and 18 DEGs (Fig. 4A). These results suggest that members of the ERF, bHLH, and MYB families may play key roles in regulating gene expression in response to CaCl₂ treatment.

Fig. 4.

Transcription factor (TF) prediction analysis revealed key TF families in Ca.²⁺-mediated regulation of cucumber sex differentiation. A Number of DEGs in the top 23 predicted TFs across 56 TF families. The ERF, bHLH, and MYB families contained the largest numbers of DEGs. B-F Expression patterns of the top five TF families, shown as heatmaps of log₂-transformed FPKM values. These TF families include: ERF (B), bHLH (C), MYB (D), C2H2 (E), and MADS-box (F)

In the ERF family, most members were predominantly downregulated across CaCl₂ treatment groups (Fig. 4B). In the bHLH family, distinct expression patterns were observed across the treatment groups. A subset of bHLH genes, including CRJ35, MYC2, and BH019, was strongly downregulated following CaCl₂ application. In contrast, several genes such as PIF7, UNE10, and FAMA showed clear induction at Ca-1W, while others, including HEC3, BH030, and BH096, were activated at Ca-2W or Ca-3W relative to CK. These expression dynamics highlight the diverse regulatory roles of bHLH genes during the Ca²⁺-mediated response (Fig. 4C). The MYB family displayed contrasting expression patterns among its members, with some genes upregulated and others downregulated following CaCl₂ treatment. Notably, MYB62_2, MYB61, and MB101 were strongly upregulated at Ca-3W (Fig. 4D). The C2H2 family showed a similar overall trend, with certain genes (ZAT11_1, ZAT10_1) downregulated and others (ZWIP6, IDD7) upregulated (Fig. 4E). The MADS − box family displayed a highly consistent expression pattern in response to CaCl₂ treatment. These genes, including AGL42, AP1_2, AP3, MADS1, MADS2_1, and MADS2_2, showed slight upregulation at Ca-1W, followed by pronounced induction at Ca-2W and Ca-3W (Fig. 4F). This uniform trend suggests a coordinated role for MADS − box TFs in mediating Ca²⁺-dependent regulatory processes, potentially contributing to developmental responses such as floral organ differentiation. These results highlight the dynamic and family-specific transcriptional regulation in response to CaCl₂ treatment, suggesting that these TFs are associated with Ca²⁺-related processes and may be linked to sex differentiation in cucumber.

Integrated transcriptome–metabolome analysis identifies key pathways involved in Ca²⁺-mediated regulation of cucumber sex differentiation

To identify key pathways involved in Ca²⁺-mediated responses in cucumber, KEGG pathway enrichment analysis was performed on DEGs and DAMs between each CaCl₂-treated group and CK. In Ca-1W, most DEGs and DAMs were enriched in biosynthesis of secondary metabolites, plant hormone signal transduction, and phenylpropanoid biosynthesis (Fig. 5A). In Ca-2W, the most enriched pathway was metabolic pathways, followed by biosynthesis of secondary metabolites and plant hormone signal transduction (Fig. 5B). Notably, Ca-3W exhibited a similar enrichment pattern to Ca-2W, with the same top three pathways (Fig. 5C). These pathways may represent key molecular and metabolic processes through which Ca²⁺ modulates hormone signaling and promotes sex differentiation in cucumber.

Fig. 5.

Correlation analysis of DEGs and DAMs following CaCl2 treatment. A-C. KEGG pathway enrichment analysis of DEGs (triangles) and DAMs (circles) in Ca-1W (A), Ca-2W (B), and Ca-3W (C) compared with CK, respectively. The x-axis (Rich factor) indicates the ratio of annotated DEGs/DAMs to the total number of genes/metabolites in the pathway. Point size corresponds to the number of DEGs/DAMs annotated to the pathway. Point color (P value) represents the significance of enrichment. D Correlation network of DEGs and DAMs. Nodes represent genes (circles) or metabolites (squares). Node color indicates degree value, with yellow representing lower degrees and red representing higher degrees. Edges indicate significant correlations (Pearson correlation, |r|> 0.85, P < 0.05). Hub genes were identified based on degree values

Next, an integrative correlation analysis of the transcriptome and metabolome was performed. Multiple genes and metabolites displayed strong positive correlations (R > 0.85). For example, CsaV3_1G007370, enriched in the plant hormone signal transduction pathway, showed strong correlations with alangilignoside D (Lchn000012) and olivil-4'-O-glucoside (Lmnn102709). Similarly, CsaV3_3G012170, a member of the AP2/ERF-ERF family, was significantly correlated with 5'-deoxyadenosine (pmb0645), chrysoeriol-5,7-di-O-glucoside (pmp000596), cordycepin (3'-deoxyadenosine) (MWSmce116), hesperetin-6-C-glucoside-7-O-glucoside (Zmxp002867), and quercetin-3-O-(2''-O-galactosyl)glucoside (mws1715). Notably, flavonoids were the most abundant metabolites in the correlation network, suggesting their potential involvement in Ca²⁺-related processes associated with cucumber sex differentiation (Fig. 5D, Table S4).

Integrated transcriptome and metabolome analyses of plant hormone signal transduction pathways in cucumber following CaCl2 treatment

Our previous integrated transcriptome-metabolome analysis revealed significant enrichment of the plant hormone signal transduction pathway, prompting further examination of DEGs and changes in hormone levels. A total of 115 genes were identified across eight hormone signaling pathways, including ethylene, ABA, GA, cytokinins, auxin, JA, salicylic acid (SA), and brassinosteroid (BR). LC–MS/MS analysis of six major plant hormones showed that ABA and GA levels were significantly elevated in all three CaCl₂ treatment groups (P < 0.05). Levels of the other hormones showed a decreasing trend following treatment, although most changes were not statistically significant (Fig. 6). In the GA signaling pathway, 19 DEGs were identified, including four G1D1-like GA receptor genes (CXE15, CXE2, CXE12, and CXE18), five genes encoding DELLA proteins (homologous to the RGA protein in Arabidopsis), and ten phytochrome interacting factor 4 (PIF4)-related genes. Among these, most PIF4-related genes, such as PIF1, PIF7, UNE10, and HEC3, showed clear induction in Ca-1W or Ca-3W compared with CK, whereas most DELLA-related genes exhibited reduced expression under CaCl₂ treatment. These changes indicate an overall activation of GA signaling following CaCl₂ treatment. In contrast, 22 DEGs were assigned to the cytokinin signaling pathway, most of which were markedly upregulated upon CaCl₂ treatment. These genes were further grouped into functional modules, with Arabidopsis response regulators (ARRs) representing the core signaling components. Extensive upregulation of the ARR-related genes was observed at Ca-1W, Ca-2W, and Ca-3W. A total of 37 DEGs were annotated in the auxin signaling pathway, including two Auxin Transporter 1 (AUX1)-related genes, six AUX/IAA-related genes, six indole-3-acetic acid-amido synthetase (GH3)-related genes, eight small auxin up RNA 36 (SAUR36)-related genes, eight transmembrane kinase (TMK) 1/4-related genes, one L-Tryptophan aminotransferase of Arabidopsis 1 (TAA1)-related gene, and two ABA-insensitive 1/2 (ABI1/2)-related genes. Among these, genes such as LAX1, LAX5, ZAR1, SAU24, and TAR2 were upregulated in Ca-1W, Ca-2W, and Ca-3W. Other hormone signaling pathways were also enriched with DEGs. A total of 15 DEGs were mapped to the JA signaling pathway, including five jasmonate ZIM-domain (JAZ)-related genes and ten MYC2-related genes. Five of the MYC2 genes exhibited higher expression in the CaCl₂-treated groups compared with CK. In addition, 5 DEGs were mapped to the ethylene signaling pathway, 8 to the ABA pathway, 12 to the SA pathway, and 10 to the BR pathway. Most of the genes in these signaling pathways showed upregulation in Ca-1W, Ca-2W, or Ca-3W. Notably, TGA genes in the SA signaling pathway and ACS genes in the ethylene pathway were strongly induced, reflecting the broad activation of hormone-mediated signaling following CaCl₂ treatment.

Fig. 6.

Integrated transcriptomic and metabolomic analysis of plant hormone signal transduction pathways following CaCl₂ treatment. The left panel demonstrates endogenous hormone levels including abscisic acid (ABA), total gibberellin (GA), zeatin, indole-3-acetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA) across treatment groups. * P < 0.05, ** P < 0.01, ns = not significant (Student’s t-test). The main panel displays hormone signal transduction pathways for eight hormones: ethylene, ABA, GA, cytokinin, auxin, JA, SA, and brassinosteroid. Colored heatmaps beneath each pathway represent the transcript abundance of corresponding genes across treatment groups. Symbols indicate regulatory mechanisms: “ + P” = phosphorylation; “ + u” = ubiquitination; “⊣” = inhibition. The right panel shows key physiological processes regulated by each hormone pathway. The top-right insert shows metabolite abundance of S-adenosylmethionine (AdoMet) and methionine (precursors in plant hormone biosynthesis) across treatment groups. Gene abbreviations in the hormone signaling pathways follow standard plant nomenclature. Full gene names are provided in Table S5

Transcriptomic analysis of genes involved in hormone biosynthesis and sex differentiation

To investigate the hormone biosynthesis metabolic processes, the expression of genes related to these processes was analyzed. DEGs associated with SA biosynthesis exhibited a consistent expression pattern, with most genes downregulated in CaCl₂-treated groups. In contrast, genes involved in the biosynthesis of other hormones, such as ethylene, ABA, GA, cytokinin, auxin, JA, and BR, were generally upregulated in one or more CaCl₂ treatment groups. For example, CsaV3_2G025850 (ACS3/1A13/ACS11-like) and CsaV3_UNG054460 (ACS7/1A17), which are associated with ethylene biosynthesis, showed significant upregulation (Fig. 7A). Correlation analysis revealed a significant negative correlation between CsaV3_UNG054460 (ACS7/1A17) and the sex-determining gene CsaV3_4G006090 (ACS2), while the correlation with CsaV3_2G025850 (ACS3/1A13/ACS11-like) was weaker (Fig. S2).

Fig. 7.

Expression patterns of DEGs in hormone biosynthesis and flower organ development. A Heatmaps showing the expression profiles of DEGs associated with hormone biosynthesis across treatment groups. These hormones include zeatin, ABA, GA, ethylene, JA, SA, auxin, and brassinosteroid. B Heatmaps showing the expression profiles of DEGs related to flower organ development across treatment groups, including gynoecium and carpel development, floral organ development, petal development, and floral whorl development. Color scales indicate relative expression

Through functional annotation of DEGs based on the GO database, a total of 44 DEGs involved in the formation of floral organs were identified. Among these, 14 were involved in gynoecium and carpel development, most of which were substantially upregulated in Ca-3W. Additionally, genes related to the development of whorl, petal, or other floral organs were also greatly upregulated in Ca-3W (Fig. 7B). This overall upregulation pattern of the genes suggests their potential roles in female flower differentiation in cucumber, highlighting the influence of CaCl₂ treatment on floral organogenesis.

Correlation analysis between CML Genes and hormone biosynthesis and signal transduction-related DEGs

To explore the roles of CML genes in cucumber sex differentiation, we performed a correlation analysis between CML genes and all enzyme genes involved in plant hormone biosynthesis and signal transduction pathways (Fig. 8). In the resulting network, CML genes were located at the center and showed strong correlations with multiple phytohormone-related enzyme genes. Among them, CML25 exhibited the most extensive connections with other enzyme genes. Several CMLs, such as CML2, CML19, CML23, CML27, CML45, and CML46, were positively correlated with CsaV3_4G006090 (ACS2/1A1C), a key enzyme gene in ethylene biosynthesis. Notably, CML25 and CML27 were positively correlated with CsaV3_2G025850 (1A13), another ethylene biosynthesis enzyme gene. In addition, CML19 was positively correlated with SA signaling and biosynthesis pathway genes, including CsaV3_4G002320, CsaV3_4G002330, CsaV3_4G002290, and CsaV3_6G039710. These results reveal correlations between CML proteins and key hormone biosynthesis and signaling genes, highlighting a possible link to cucumber sex differentiation.

Fig. 8.

Correlation network of CML genes with hormone transduction and biosynthesis genes. Nodes represent CML genes (circles) or genes involved in hormone transduction and biosynthesis (squares). Edges indicate significant correlations (Pearson correlation, |r|> 0.85, P < 0.05). Node size reflects degree value, highlighting hub genes or key CMLs within the network

WGCNA of DEGs in response to CaCl₂ treatment

To identify key genes involved in cucumber sex differentiation under CaCl₂ treatment, we performed WGCNA on the transcriptome data. Gene expression correlations were calculated to construct a weighted co-expression network, and genes with highly similar expression patterns were grouped into 15 co-expression modules. Module–trait correlation analysis revealed that six modules, including turquoise, blue, pink, purple, green-yellow, and brown, were strongly associated with the Ca-3W group (Fig. S3 A), suggesting their involvement in female flower differentiation under CaCl₂ treatment. As the turquoise module contained the largest number of genes (3,160), a network was constructed to visualize gene interactions. Within the network, hub genes, including CsaV3_1G044720 (GPT2), KN14U CsaV3_1G039610 (KN14U), and CsaV3_7G026660 (B561C), were identified (Fig. S3 B). We then annotated the top six hub genes from the aforementioned six modules (turquoise, blue, pink, purple, greenyellow, and brown). In the turquoise module, CsaV3_6G015510 was annotated as ERF3, belonging to the AP2/ERF-ERF TF family, involved in plant hormone signal transduction. In the blue module, CsaV3_3G023550 (IAA26), CsaV3_6G052850 (RITF1), and CsaV3_1G006220 (AP1) belong to the AUX/IAA, PLATZ, and MADS-box TF families, respectively. In the brown module, CsaV3_4G011810 was annotated as a cellulose synthase catalytic subunit, playing an important role in plant growth, development, and resistance. In the purple module, CsaV3_1G000200 (LCB2A) plays an essential role in male gametophyte development in Arabidopsis (Table S6). Pearson correlation analysis between hub genes from the six modules and the number of female flowers revealed significant correlations, such as CsaV3_4G011810 (CESA3), CsaV3_3G048900 (HDG5), CsaV3_6G015510 (ERF3), and CsaV3_7G026660 (B561C) (Fig. S3C). Further KEGG enrichment analysis of the six modules was subsequently performed. The results showed that the greenyellow module was significantly enriched in nucleocytoplasmic transport and RNA surveillance pathways, whereas the brown module was enriched in the autophagy pathway (Fig. S3D).

qRT-PCR verification

To validate the transcriptome data, qRT-PCR analysis was performed on CML genes, floral organ formation–related genes (Fig. 9A-B), and genes involved in hormone signal transduction, flavonoid biosynthesis, and cysteine and methionine metabolism (Fig. S4A-B). The qRT-PCR results were largely consistent with the transcriptome data, showing similar expression trends.

Fig. 9.

Validation of gene expression by quantitative reverse transcription PCR (qRT-PCR). Bar plots (A) and heatmaps (B) showing relative expression levels of 10 CML genes and 10 genes involved in floral organ formation across treatment groups. The color scale represents the relative expression of genes

Discussion

Sex determination in cucumber is not controlled by a single gene or linear pathway. Rather, it is governed by a complex, multidimensional network integrating hormonal signaling, developmental programs, metabolic pathways, cellular architecture, and environmental cues. This networked regulation enables the plant to modulate the formation of male, female, or hermaphrodite flowers in a flexible, context-dependent manner [1, 38–41]. Han et al. [42] further showed that sex determination is an intricate network centered on CsARF3-mediated auxin signaling: CsARF3 promotes female flower development by activating the meristem maintenance gene CsSTM and repressing the gynoecious gene CsWIP1, while auxin and ethylene signaling engage in bidirectional crosstalk. Together, this network integrates multiple genes, hormone interactions, and feedback loops to regulate cucumber floral sex determination. In this study, the androecious cucumber line ‘Banna 31–10’ was used as experimental material, and CaCl₂ was applied on cucumber seedlings. We demonstrated that exogenous CaCl₂ treatment promotes female flower formation in cucumber and triggers extensive transcriptional and metabolic reprogramming. Through integrative analyses of DEGs, DAMs, CML–hormone correlation networks, and WGCNA modules, we uncovered a Ca²⁺-associated regulatory landscape involving hormone biosynthesis and signaling, TF expression patterns, metabolite accumulation, and floral organ development.

Integrated KEGG enrichment analysis of DEGs and DAMs revealed that both genes and metabolites were primarily enriched in the biosynthesis of secondary metabolites, plant hormone signal transduction, and phenylpropanoid biosynthesis. The phenylpropanoid biosynthesis pathway is a major plant secondary metabolic route that converts phenylalanine into a wide range of compounds, including flavonoids, lignin, and other phenolic metabolites [43, 44]. Although phenylpropanoid synthesis has not been directly linked to floral organ development, the identification of 230 flavonoids as DAMs in this study provides indirect evidence that this pathway may be closely associated with flower organ development. However, the biological functions of DEGs highlighted in the heatmap (Fig. 2C) remain to be validated, and future studies employing gene overexpression and silencing approaches will be essential to clarify their regulatory roles.

Subsequent analysis of hormone signal transduction pathways showed that CaCl₂ treatment broadly altered multiple pathways, including GA, cytokinin, ABA, SA, ethylene, JA, BR, and auxin. This alteration was reflected by the upregulation of key genes such as ACS (ethylene), ARR (cytokinin), and TGA (SA). These findings reveal associations between Ca²⁺-responsive processes and hormone pathway dynamics that may relate to cucumber sex differentiation and floral organ development. Consistently, our transcriptomic analysis of hormone biosynthesis pathways revealed upregulation of ACS genes involved in ethylene biosynthesis. In cucumber, ethylene is a key factor associated with sex differentiation, and several ACS genes have been reported as important determinants of floral organ development [45]. In our study, CsaV3_2G025850 (ACS3/1A13/ACS11-like) was strongly upregulated under CaCl₂ treatment, consistent with previous findings. ACS11 in cucumber is specifically expressed in developing female flowers, where it collaborates with ACC oxidase 2 (ACO2) to produce ethylene, thereby repressing male flower development and promoting carpel/female flower formation [46, 47]. A loss-of-function mutation (S301F) in ACS11 abolishes its enzymatic activity, resulting in an androecious phenotype [48, 49]. Interestingly, in addition to the well-characterized cucumber sex-determining gene ACS11, we observed a marked upregulation of CsaV3_UNG054460 (ACS7/1A17) under CaCl₂ treatment. In Arabidopsis, ACS7 is a type‑III ACC synthase mainly implicated in root gravitropism, vascular cambium activity, leaf senescence, and stress responses [43, 44, 50]. Currently very limited evidence links CsACS7 to floral organ development or sex differentiation. In Fragaria vesca (strawberry), ACS7 RNAi plants failed to flower, whereas overexpression slowed growth, suggesting a role in general flower development [51]. Although we observed an expression association between CsaV3_UNG054460 (ACS7/1A17) and CsaV3_4G006090 (ACS2), this observation should be interpreted with caution. Future functional studies will be necessary to clarify the precise role of CsaV3_UNG054460 (ACS7/1A17) in cucumber development and sex differentiation.

Our analysis identified ERF, bHLH, MYB, C2H2, and MADS − box TFs as most responsive to CaCl₂ treatment. ERF genes were predominantly downregulated following CaCl₂ treatment, suggesting a general suppression of certain ethylene-responsive transcriptional programs. In contrast, a few ERF members, such as PPAF2, DREB3_1/2, and ERF39 were upregulated, indicating that Ca²⁺ signaling may selectively activate specific ERFs while repressing others. Studies in banana show that DREB TFs, including MusaDREB1G, function primarily as regulators of abiotic stress responses and fruit ripening, where they modulate gene activation, hormone signaling and reactive oxygen species (ROS) scavenging [52, 53]. In addition, TFs from other families, such as NAC (MusaSNAC1 and MpSNAC67) and MYB (MusaMYB31), have also been reported in banana to regulate stress-responsive, phytohormone-related, or metabolic pathways [54–58]. Although these regulators were not the primary focus of the present study, these findings from another major fruit crop further support the broader role of TFs in coordinating hormone-associated and developmentally relevant regulatory networks.

Pawełkowicz et al. identified MADS‑box TFs as one of the most crucial regulators in cucumber sex determination, highlighting their roles in floral organ specification and development [1, 40]. Consistently, our study found that CaCl₂ treatment induced substantial upregulation of MADS-box genes. MADS‑box genes are well-established regulators of floral organ identity and development, forming the core of the so-called ABCDE model [59–61]. Beyond organ specification, MADS‑box TFs also influence flowering time, meristem identity, and the transition from vegetative to reproductive growth [62–64]. In cucumber, phylogenetic analysis indicated that most MADS‑box genes are conserved and likely perform similar roles as their counterparts in other plant species [65]. Moreover, the MADS‑box gene CsSHP is specifically enriched in reproductive organs and interacts with SEP proteins. It is also suggested to be involved in fruit maturation through the ABA pathway and may contribute to floral organ specification [66]. In this study, seven A-class genes (MADS1, MADS2, MADS3, MADS4, ERAF17 (MADS6), AP1, AP3) of the MADS-box family were highly expressed at Ca-2W and Ca-3W. ERAF17 has been shown to be rapidly induced by the ethylene-releasing compound ethephon and maintained throughout female flower development, suggesting that it mediates ethylene-driven female flower formation at the apices [67]. The coordinated induction of multiple MADS‑box genes in our study reveals potential links to female flower formation. These changes may reflect alterations in MADS protein complexes that correlate with floral organ development toward pistil/carpel identity [68]. Notably, AP1-1 and AP1-2, two key MADS-box family TFs, were markedly upregulated following CaCl₂ treatment. Moreover, CsaV3_1G006220 (AP1) was identified as a hub gene within the blue module in WGCNA. The combination of dynamic upregulation and hub-gene status highlights AP1 as a potential key regulator mediating Ca²⁺-dependent floral organ differentiation, possibly contributing to pistil/carpel development. Currently, AP1 is well-known for regulating floral meristem identity and organ specification in Arabidopsis [69], Alejandra [70]. However, its role in cucumber remains largely uncharacterized. Zhou et al. reported that the AP1-like MADS-box gene CsMADS09 is predominantly expressed in reproductive tissues, influences flowering time and leaf development when ectopically expressed in Arabidopsis, and likely plays a key role in regulating flower development in cucumber [71]. Nevertheless, the specific function of AP1 in cucumber requires further investigation. The interaction between Ca²⁺ signaling and MADS-box has been identified in a recent study in apple (Malus domestica), which demonstrates that Ca²⁺ suppresses ethylene biosynthesis by promoting MdCDPK7-mediated phosphorylation of the MADS-box TF MdMADS5 and the ethylene biosynthesis enzyme MdACO1, thereby delaying fruit ripening [72]. While analogous interactions have not yet been reported in cucumber, the dynamic expression of multiple MADS-box genes following CaCl₂ treatment in our study suggests that ca²⁺ signaling may modulate MADS-box activity during female flower formation. Future functional studies, such as yeast two-hybrid analysis or co-immunoprecipitation, will be needed to explore this regulatory link in Ca²⁺-mediated floral organ differentiation.

CML proteins constitute a major class of plant-specific Ca²⁺ sensors that decode intracellular Ca²⁺ signatures and translate them into downstream physiological responses [48, 49]. Extensive studies have shown that CMLs participate in diverse developmental and stress-related processes by modulating hormone biosynthesis and signaling. For instance, Arabidopsis CML37 and CML42 regulate jasmonic acid–dependent defense responses [28], and CML24 modulates ABA sensitivity [73]. These findings highlight associations between CMLs and hormone-related pathways in the context of CaCl₂ treatment. In our study, correlation network analysis revealed that multiple CsCML genes were strongly associated with key enzyme genes in hormone biosynthesis and signaling pathways. Specifically, CML2, CML19, CML23, CML27, CML45, and CML46 showed strong positive correlations with ACS2/1A1C and 1A13, major ethylene biosynthetic enzymes that are essential for female flower determination in cucumber. Additionally, CML19 was correlated with several SA pathway genes, indicating potential involvement in SA-mediated reproductive development. At the same time, the hormone pathways associated with these CMLs can also regulate CML expression, implying potential feedback interactions. In papaya, multiple CML genes were found to be strongly regulated by ethylene [74], while in Arabidopsis, CML43 functions as a Ca²⁺ sensor whose expression and protein accumulation are induced by SA [75]. So far, 44 CsCMLs family members have been identified in the cucumber genome, and CsCML genes exhibited relatively elevated expression in flowers, especially CsCML31, CsCML3, and CsCML13 [76]. Among CsCMLs, CsCML25 emerged as a hub gene with the most correlations to hormone-related enzymes in our network. In Arabidopsis, CML25 regulates pollen germination, tube elongation, and seed setting by modulating Ca²⁺-dependent K⁺ influx in pollen grains and tubes [77], with the regulation of pollen tube extension within the pistil directly affecting pistil fertility [78]. The overexpression of CML25-like in cucumber led to high expression in young leaves and female flowers, promoted fruit development, and dramatically increased the parthenocarpy rate to 98.25%, 86% higher than the control [29]. Collectively, these findings highlight associations between CsCMLs, CaCl₂–related responses, and hormone pathways, which may be linked to sex differentiation and reproductive development.

Ca²⁺ acts as a universal second messenger regulating diverse developmental processes across plant species, including reproductive and floral development [79–81]. In Arabidopsis, dynamic cytosolic Ca²⁺ signals occur throughout double fertilization, with distinct Ca²⁺ signatures in synergid, egg, and central cells coordinating pollen tube reception, sperm release, and successful fertilization [82]. A recent study in tomato demonstrated that Ca²⁺ signaling regulates drought-induced flower drop through the SlCBL11–SlCIPK10–SlLHP1b module, linking ca²⁺ signaling to epigenetic repression of auxin biosynthesis in stamens and subsequent reproductive organ abscission [83]. Direct evidence linking Ca²⁺ signaling to sex determination remains limited; however, in pumpkin (Cucurbita maxima), CmaCPK4 was shown to regulate sex determination by interacting with and phosphorylating ethylene biosynthesis enzymes (CmaACS5 and CmaACS7), thereby increasing ethylene production and promoting female flower formation [84]. Collectively, these cross-species observations, together with our transcriptomic and metabolomic data in cucumber, suggest that Ca²⁺-associated networks integrate hormonal signals, transcriptional changes, and metabolic reprogramming to modulate sex differentiation and reproductive flexibility.

Despite the integrative analyses performed in this study, several limitations should be acknowledged. In cucumber, floral sex differentiation is initiated during early shoot apex development, prior to visible flower formation [85]. Therefore, sampling at the seedling stage enables the capture of the initial regulatory shifts that determine sex expression; however, SAM samples used for transcriptomic and metabolomic analyses may contain non-reproductive or vegetative tissues, potentially diluting or obscuring flower-specific signals. Future studies targeting floral tissues will be necessary to clarify how these early regulatory events translate into morphological outcomes and to minimize potential interference from non-reproductive tissues. Although multi-omics integration revealed potential correlations among genes and metabolites, these associations are primarily correlative and do not establish direct causal relationships. Functional validation will be necessary to confirm potential mechanistic links. Overall, our findings extend the current framework of cucumber sex determination, provide a foundation for future functional investigations, and offer potential strategies for manipulating floral sex in cucumber breeding and cultivation.

Authors’ contributions

Can Wang & Xiang Guo: Formal analysis & Writing – original draft; Jinfang Cha & Wenlin Zhao: Methodology & Resources; Liu Huang & Kai Zhao: Investigation & Data curation; Yingnan Tian: Supervision & Writing – review and editing; Junqiang Xu: Conceptualization, funding acquisition, Writing – review and editing.

Funding

This study was supported by Yunnan Fundamental Research Projects (202301AT070502), Major Science and Technology Projects in Yunnan Province (202402AE090012), and Xingdian Talent Support Program of Yunnan Province (XDYC-QNRC- 2023–0428).

Data availability

The sequencing data generated in this study have been deposited in the NCBI database under BioProject accession number PRJNA1298966 and are publicly available at: https://www.ncbi.nlm.nih.gov/search/all/?term=PRJNA1298966.

Declarations

Ethics approval and consent to participate

All experimental research involving plants, including the collection of plant material, was conducted in accordance with institutional, national, and international guidelines. Field studies were carried out following local legislation, and all necessary permissions and/or licenses for the collection and use of plant material were obtained from the relevant authorities.

Competing interests

The authors declare no competing interests.