Comparative Transcriptomics Reveals Synergistic Control of Cucumber Sex Determination by Ethylene Signaling and Epigenetic Regulation

doi:10.13560/j.cnki.biotech.bull.1985.2025-0571

Abstract

Abstract:

Objective To identify the key genes and regulatory pathways that control sex determination in cucumber (Cucumis sativus L.), with the aim of providing a theoretical basis for breeding high-yield cucumber varieties and laying a foundation for understanding the mechanisms of sex determination in dicotyledonous plants. Method Near-isogenic lines of cucumber, the gynoecious line X8g and the monoecious line X8, were used as materials. Transcriptome sequencing (RNA-Seq) analysis was conducted on the apical meristems of flower buds at the four-leaf-one-heart stage. An integrated strategy combining differential expression analysis, principal component analysis (PCA), and variance decomposition analysis was employed to screen for key genes. OrthoFinder was used to identify orthologous genes between cucumber and Arabidopsis thaliana to perform a cross-species comparative transcriptomic analysis, investigating the conservation and specificity of the regulatory networks. The function of the core candidate gene, CsACO2, was validated using Virus-Induced Gene Silencing (VIGS), and its silencing efficiency and specificity were assessed by qPCR. The role of epigenetic regulation was verified by treating the gynoecious line with the DNA methylation inhibitor 5-azacytidine. This study investigated key genes and pathways in cucumber sex determination using RNA-Seq analysis of gynoecious and monoecious lines. Result A total of 197 differentially expressed genes were identified between the gynoecious and monoecious lines, with 43 being upregulated and 154 downregulated. These genes were significantly enriched in pathways such as floral organ morphogenesis and α-linolenic acid (a precursor for jasmonic acid synthesis) metabolism. The multi-dimensional analysis identified a set of core candidate genes, including the known key ethylene synthesis gene CsACO2 and newly discovered MADS-box transcription factors like AGL6 and MADS4. The cross-species comparative analysis showed a weak overall correlation in expression patterns of orthologous genes but revealed the potential importance of target gene sets of the ethylene response factor EIN3, the NAC053 transcription factor, and H3K27me3 epigenetic modification in regulating cucumber sex. Functional validation experiments confirmed that the specific silencing of CsACO2 induced the formation of male and bisexual flowers in the gynoecious line. Similarly, treatment with 5-azacytidine also caused the gynoecious line to produce male flowers. Conclusion This study systematically reveals that cucumber sex determination is governed by a complex molecular network dominated by the ethylene signaling pathway, potentially involving jasmonic acid metabolism, various transcription factors, and epigenetic modifications.

Key words: Cucumis sativus L., sex determination, comparative transcriptomics, ethylene signaling pathway, CsACO2, epigenetic modification, virus-induced gene silencing (VIGS), Arabidopsis thaliana

YANG Zong-hui, LI Li-bin, MENG Zhao-juan, GAO Tian, ZHU Li-xia, DU Hai-mei, DONG Wei-wei, CAO Qi-wei. Comparative Transcriptomics Reveals Synergistic Control of Cucumber Sex Determination by Ethylene Signaling and Epigenetic Regulation[J]. Biotechnology Bulletin, 2025, 41(12): 139-155.

Figures/Tables 11

Table 1 Basic statistics of sequencing data

样本编号 Sample ID	清洁读数 Clean reads	清洁碱基数 Clean bases （bp）	GC 含量 GC content （%）	Q30 碱基比例 Q30 （%）
T01	25 139 532	7 511 087 362	44.41	93.75
T02	23 678 722	7 068 974 554	44.57	93.75
T03	29 026 750	8 662 561 328	44.31	94.09
T04	31 798 263	9 491 036 146	44.43	93.77
T05	26 566 141	7 941 970 238	44.24	93.74
T06	28 078 078	8 390 027 920	44.29	93.69

Table 2 Statistics of mapping results

样本编号 Sample ID	总读数 Total reads	比对读数 Mapped reads	唯一比对读数 Uniquely mapped reads	多重比对读数 Multi-mapped reads	正链比对读数 Mapped reads on+strand	负链比对读数 Mapped reads on-strand
T01	50 279 064	48 741 549 （96.94%）	47 020 390 （93.52%）	1 721 159 （3.42%）	24 142 446	24 233 782
T02	47 357 444	45 842 026 （96.80%）	44 215 419 （93.37%）	1 626 607 （3.43%）	22 699 707	22 792 183
T03	58 053 500	56 296 505 （96.97%）	54 317 173 （93.56%）	1 979 332 （3.41%）	27 877 852	27 989 891
T04	63 596 526	61 603 424 （96.87%）	59 470 279 （93.51%）	2 133 145 （3.35%）	30 511 057	30 629 863
T05	53 132 282	51 558 779 （97.04%）	49 751 449 （93.64%）	1 807 330 （3.40%）	25 547 018	25 635 938
T06	56 156 156	54 471 955 （97.00%）	52 558 943 （93.59%）	1 913 012 （3.41%）	26 972 777	27 079 418

Fig. 1 Transcriptome expression pattern analysis of gynoecious and monoecious cucumber linesA: Principal component analysis (PCA) plot showing overall expression differences among samples. PC1 and PC2 explain 38.4% and 23.6% of expression variation, respectively. Colors indicate different genotypes: gynoecious (red) and monoecious (blue). B: Volcano plot showing the distribution of differentially expressed genes. Red and blue dots represent significantly up-regulated (43) and down-regulated (154) genes (|log2FC|>=1, FDR<0.01). Highlighted are the genes with a fold change in expression exceeding 3 times. C: Distribution of gene loadings on PC1 and PC2. Dot colors indicate differential expression status. Key genes with high PC1 loadings are highlighted. D: Variance decomposition analysis of gene expression. The X-axis shows the distribution of genotype variance proportions, while the Y-axis displays the logarithmic ratio of genotype variance to residuals. Highlighted are the genes with a genotype variance proportion exceeding 0.99. E: Venn diagram showing the overlap among genes identified by three screening methods (differential expression analysis, principal component analysis, and variance decomposition). Numbers indicate gene counts and percentages in each region

Table 3 Key genes identified by three screening methods and their expression characteristics

基因编号 Gene ID	基因名称 Gene name	log₂倍数变化 log₂FC	表达调控 Regulation	PC1载荷值 PC1 loading	基因型方差占比 Fraction of variance across genotypes
CsaV3_2G032190	--	-1.23	下调	-0.020	0.927
CsaV3_3G040160	--	-1.74	下调	-0.020	0.931
CsaV3_4G028010	--	-2.43	下调	-0.032	0.998
CsaV3_4G037760	SPL14	1.26	上调	0.049	0.938
CsaV3_5G024890	--	-5.41	下调	-0.203	0.994
CsaV3_6G006010	AGL6	-1.98	下调	-0.028	0.999
CsaV3_6G008200	MADS4	-2.90	下调	-0.027	0.988
CsaV3_6G048630	CsACO2	-1.20	下调	-0.050	0.991
CsaV3_6G051850	--	2.81	上调	0.033	0.964

Fig. 2 Functional enrichment analysis reveals key metabolic pathways and biological processesA: KEGG pathway enrichment analysis results. Bubble size indicates the number of enriched genes, and color intensity indicates the significance level (adjusted P-value). B: GO functional enrichment analysis results. Bubble size indicates gene count, and color indicates adjusted P-value. C: Comparison of KEGG pathway enrichment based on different screening strategies. Heatmap colors indicate enrichment scores (NES), with yellow indicating positive enrichment and blue indicating negative enrichment. The size of a dot indicates the significance level [-log10(P.adjust)]. D: Comparison of GO functional enrichment for gene sets obtained from different selection strategies. The colors in the heatmap indicate the enrichment score (NES), with yellow indicating positive enrichment and blue indicating negative enrichment. The size of a dot indicates the significance level [-log10(P.adjust)]. FC: Differential expression analysis; PCA: principal component analysis; VD: variance decomposition

Fig. 3 Comparative analysis of orthologus gene expression patterns between cucumber and ArabidopsisA: Scatter plot showing expression changes of orthologous genes between cucumber and Arabidopsis. X-axis and Y-axis indicate log2FC values in cucumber and Arabidopsis, respectively. Colors indicate different expression patterns. Key genes are labeled by gene names. B: GO functional enrichment analysis of cucumber-specific differentially expressed genes. Bubble size indicates gene count, and color intensity indicates significance level (adjusted P-value)

Fig. 4 Expression heatmap analysis of key genes in cucumber and ArabidopsisThe heatmap displays the expression patterns of 101 differentially expressed genes with gene symbol annotations in cucumber and Arabidopsis. Each row indicates a gene, and the colors indicate changes in gene expression levels: yellow indicates upregulated expression, while blue indicates downregulated expression. The genes are clustered based on their expression patterns. The annotation bar on the right shows the species origin and genotype. The FPKM values of each row (i.e., each gene) are normalized to Z-score scale

Fig. 5 Functional analysis of species- and genotype-specific genesA: Vriance distribution plot of gene expression. The X-axis indicates the fraction of variance across species, while the Y-axis indicates the fraction of variance across genotypes. The color of the points indicates different categories: high genotype variance proportion (red), high species variance proportion (green), and others (gray). B: KEGG pathway enrichment analysis results for genes regulated by genotype effects. The size of the bubbles indicates the number of genes, and the color intensity indicates the adjusted P-value. The X-axis indicates the enrichment fold change. C: GO functional enrichment analysis results for genes regulated by genotype effects. The size of the bubbles indicates the number of genes, and the color intensity indicates the adjusted P-value. The X-axis indicates the enrichment fold change. D: GSEA enrichment analysis results for genes regulated by genotype effects. The size of the bubbles indicates the number of genes set, the color indicates the adjusted P-value, and the X-axis indicates the normalized enrichment score (NES)

Fig. 6 Gene set enrichment analysis of PlantGSADA: Enrichment analysis results for PlantGSAD gene sets regulated by genotype effects. The lollipop chart displays significantly enriched PlantGSAD gene sets, where the size of a bubble indicates the size of the gene sets, the color intensity indicates the adjusted P-value, and the X-axis indicates the normalized enrichment score. B: Enrichment analysis results for the EIN3 target gene set. The upper panel shows the running enrichment score, while the lower panel displays the distribution of genes in the ranking of genotype variance proportions. C: Enrichment analysis results for the NAC053 target gene set. It similarly displays the running enrichment score and the gene distribution plot

Fig. 7 VIGS analysis of the CsACO2 gene in gynoecious cucumberA: Negative control (pTRSV2-empty vector), in which the infected gynoecious cucumber (X8g) plant produces only normal female flowers. B: Positive control (pTRSV2-PDS), in which the leaves of the infected X8g plant show significant photobleaching, indicating the VIGS system is working. C: Silencing of CsACO2 (pTRSV2-CsACO2) induces the formation of male flowers on the infected X8g plant. D: Silencing of CsACO2 (pTRSV2-CsACO2) induces the formation of bisexual flowers on the infected X8g plant. Scale bar=1 cm. E‒I: RT-qPCR results showing significant downregulation of CsACO2 gene in silenced plants, while other ACO family members (CsACO1, CsACO3, CsACO4, CsACO5) showed no significant changes. CK: Empty vector control; V1, V2: two independent CsACO2-silenced plants. Bar charts show relative expression levels (mean±SD), ***P<0.001

Fig. 8 Chemical treatment of gynoecious cucumber with the DNA methylation inhibitor 5-azacytidinePhenotypic difference between the 5-azacytidine treatment group (5-az) and control group (H2O). The control gynoecious X8g plant grows normally and produces female flowers, whereas the 5-az-treated plant produces male flowers. Scale bar=10 cm

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