Temporal Transcriptome Analysis Reveals Core Pathways and Orphan Gene EARLY FLOWERING 1 Regulating Floral Transition in Chinese Cabbage

Abstract

The floral transition in Chinese cabbage (Brassica rapa ssp. pekinensis) is governed by a complex interplay of gene expression and hormonal regulation. Temporal transcriptome profiling was conducted across three developmental stages: pre-bolting (PBS), bolting (BS), and flowering stages (FS), to investigate the underlying molecular mechanisms. A total of 7092 differentially expressed genes (DEGs) were identified, exhibiting distinct expression trajectories during the transition. Moreover, functional enrichment analyses revealed strong associations with plant hormone signaling, MAPK pathways, and developmental regulation processes. Key flowering-related genes, such as BrFLM, BrAP2, BrFD, BrFT, and BrSOC1s displayed antagonistic expression patterns. Hormonal pathways involving auxin, ABA, ET, BR, GA, JA, CK, and SA showed stage-dependent modulation. Further, orphan genes (OGs), especially EARLY FLOWERING 1 (EF1), showed significant upregulation during the transition, which exhibited 1.84-fold and 1.93-fold increases at BS and FS compared to PBS, respectively (p < 0.05). Functional validation through EF1 overexpression (EF1OE) in Arabidopsis consistently promoted early flowering. The expression levels of AtFT and AtSOC1 were significantly upregulated in EF1OE lines compared to wild-type (WT) plants. The findings contribute to understanding the coordinated genetic and hormonal events driving floral development in Chinese cabbage, suggesting EF1 as a candidate for bolting resistance breeding. This work also expands the existing regulatory framework through the successful integration of OGs into the complex floral induction system of Brassica crops.

Keywords: Chinese cabbage; EARLY FLOWERING 1; MAPK signaling; floral transition; hormone signaling; orphan genes; transcriptome profiling.

Figure 1

Temporal transcriptome dynamics during floral transition in Chinese cabbage. (A) Principal component analysis (PCA) plot. (B) Correlation analysis across samples. Red indicates positive correlation; blue indicates negative correlation. (C) The count of significantly upregulated and downregulated genes across different groups. (D) Comparative Venn analysis of DEGs. Blue represents BS_vs_PBS group; yellow represents FS_vs_PBS group; pink represents FS_vs_BS group. (E) Heat map analysis. Red represents upregulated genes; green represents downregulated genes. PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively.

Figure 2

Go classification analysis of DEGs during floral transition in Chinese cabbage. (A) BS_vs_PBS group. (B) FS_vs_PBS group. (C) FS_vs_BS group. BP, CC, and MF indicate biological process, cellular component, and molecular function, respectively. The orange and blue bar charts represent upregulated and downregulated genes, respectively. PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively. The horizontal axis of BP terms with ellipsis represents the biological process involved in interspecies interaction between organisms.

Figure 3

KEGG pathway enrichment analysis of DEGs during floral transition in Chinese cabbage. (A) BS_vs_PBS group. (B) FS_vs_PBS group. (C) FS_vs_BS group. The green, purple, pink, blue, and yellow bar charts respectively represent cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems. PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively.

Figure 4

Expression patterns of core flowering pathway DEGs. (A) Heatmap analysis. Each column denotes an independent biological sample, while each row represents a unique gene transcript. Red represents upregulated genes; green represents downregulated genes. (B–G) qRT-PCR validation. Different lowercase letters denote statistically significant differences (p < 0.05, one-way ANOVA). PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively.

Figure 5

Heatmap analysis of the expression profiles of DEGs involved in plant hormone signal transduction. (A) Auxin signaling pathway. (B) ABA signaling pathway. (C) ET signaling pathway. (D) BR signaling pathway. (E) GA signaling pathway. (F) JA signaling pathway. (G) CK signaling pathway. (H) SA signaling pathway. Red represents upregulated genes; green represents downregulated genes. PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively.

Figure 6

qRT-PCR validation of DEGs involved in plant hormone signal transduction. Relative expression of (A) BrSAUR21, (B) BrPYL6, (C) BrERS1, (D) BrBZR2, (E) BrGA20ox2, (F) BrJAZ10, (G) BrCKX3, (H) BrLOG8, and (I) BrICS1. Different lowercase letters denote statistically significant differences (p < 0.05, one-way ANOVA). PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively.

Figure 7

Expression patterns of DEGs in the MAPK signaling pathway. (A) Heatmap analysis. Each column denotes an independent biological sample, while each row represents a unique gene transcript. Red represents upregulated genes; green represents downregulated genes. (B–D) qRT-PCR validation. Different lowercase letters denote statistically significant differences (p < 0.05, one-way ANOVA). PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively.

Figure 8

Heatmap analysis of the expression profiles of DEGs involved in the OGs pathway. (A) BrOGs signaling pathway. (B) BSGs signaling pathway. (C) CSGs signaling pathway. Red represents upregulated genes; green represents downregulated genes. PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively.

Figure 9

qRT-PCR validation of DEGs involved in the OGs pathway. Relative expression of (A) EF1, (B) BraA05003290, (C) BraA02002116, (D) BraA10001780, (E) BraA01002024, and (F) BraA02003961. Different lowercase letters denote statistically significant differences (p < 0.05, one-way ANOVA). PBS, BS, and FS represent the pre-bolting stage, bolting stage, and flowering stage, respectively.

Figure 10

Phenotypic characterization of Arabidopsis EF1OE lines. (A) Early flowering phenotype of EF1OE transgenic plants. Scale bar = 2 cm. (B) Flowering time. (C) Rosette leaf number. (D) Rosette radius. (E) Plant height. (F) Silique length. (G) Seed number. (H,I) Expression analysis of AtFT and AtSOC1. Data are means ± SE of three independent measurements. Significant differences were identified when comparing WT and EF1OE plants using Student’s t-tests (ns, p > 0.05, ** p < 0.01, *** p < 0.001).