Unveiling the molecular mechanism of sepal curvature in Dendrobium Section Spatulata through full-length transcriptome and RNA-seq analysis

Abstract

Introduction: Orchids are renowned for their intricate floral structures, where sepals and petals contribute significantly to ornamental value and pollinator attraction. In Dendrobium Section Spatulata, the distinctive curvature of these floral organs enhances both aesthetic appeal and pollination efficiency. However, the molecular and cellular mechanisms underlying this trait remain poorly understood.

Methods: Morphological characteristics of five hybrids were analyzed, with a particular focus on hybrid H5, which exhibits pronounced sepal curling. Full-length transcriptomic sequencing was employed to assemble a reference transcriptome, while RNA-seq identified differentially expressed genes (DEGs) between sepals and petals. Gene ontology and pathway enrichment analyses were conducted to uncover biological processes associated with sepal curvature. Cytological microscopy was used to examine cell size and number, and quantitative real-time PCR (qRT-PCR) was performed to validate transcriptomic findings.

Results: The reference transcriptome contained 94,258 non-redundant transcripts, and RNA-seq identified 821 DEGs between sepals and petals, with 72.8% of these upregulated in sepals. Enrichment analysis revealed the significant involvement of DEGs in cytokinesis, cytoskeletal organization, and energy metabolism. Notably, myosin II filament organization was implicated in generating the mechanical forces responsible for curling, while metabolic pathways provided the energy necessary for these developmental processes. Cytological observations showed that the upper cell layers of the sepal were smaller and more numerous than the lower layers, indicating that differential cell growth contributes to sepal curvature. qRT-PCR analysis validated the differential expression of selected genes, supporting the transcriptomic findings.

Discussion: The interplay of cellular mechanics, cytoskeletal dynamics, and metabolic regulation is crucial in shaping sepal morphology. Future studies involving gene knockdown or overexpression experiments are recommended to validate the roles of specific genes in processes such as actin organization and myosin activity. Such work would provide deeper insights into the contributions of cytoskeletal dynamics and mechanical force generation to sepal morphogenesis.

Keywords: Dendrobium; cytokinesis; floral development; myosin filament; sepal curvature; transcriptomics.

Figure 1

Morphological characterizations of plant and flower organs. (A) Morphological phenotypes of flowers from five orchid hybrids (H1, H2, H3, H4, and H5) from Den. Sect. Spatulata. The petals and sepals used for the curvation rate calculations were numbered and indicated on H5. (B) Comparison of the curvation rates for petals and sepals among the five hybrids. The curvation rate was determined by dividing the natural length by the flat length of the petals or sepals. Significant differences between hybrids for each organ were assessed using multiple mean comparisons. Different letters above the bars within each organ indicate statistical significance (P < 0.05, t-test). (C) Morphological phenotypes of H5 flower organs at various flowering stages. (D) Image of the H5 plant at the late flowering stage.

Figure 2

Read length distribution of the full-length transcriptome sequences. (A) Distribution of the CCS read lengths. (B) Distribution of the FLNC read lengths. (C) Distribution of the read lengths for the high-quality (HQ) consensus isoforms.

Figure 3

Analysis of transcripts and proteins derived from the full-length transcriptome. (A) Transcript completeness assessment using BUSCO software. (B) Distribution of nr homologous species annotated with non-redundant transcript sequences. (C) SSR density for eight types of predicted SSRs, identified using MISA software. (D) Length distribution of the predicted complete proteins.

Figure 4

Gene expression distribution and PCA analysis of all samples. (A) Expression distribution across all samples. The x-axis represents the 24 samples used in this study, and the y-axis represents gene expression normalized by log₂(TPM+1). (B) Principal component analysis (PCA) of all the samples, with sepals and petals represented by blue and yellow, respectively.

Figure 5

Volcano plot and heatmap of differentially expressed genes (DEGs). (A) Volcano plot showing downregulated (blue), normal (grey), and upregulated (red) genes. (B) Heatmap of 223 downregulated and 598 upregulated DEGs. TPM values were scaled by row using the “pheatmap” package in R. Sepal and petal samples are indicated by the cyan and red horizontal bars above the heatmap. The four gene clusters are marked on the right side of the heatmap.

Figure 6

GO functional enrichment analysis of the DEGs. (A) Dot plot of the significantly enriched GO terms for all the DEGs. (B) Hierarchical tree of significant GO terms. The GO terms with an adjusted P-value < 0.05 were considered significant. For categories with more than 15 significant terms, only the top 15 terms in each category (BP, Biological Process; CC, Cellular Component; MF, Molecular Function) were plotted.

Figure 7

Network visualization of GO terms and their enriched genes. (A) Network illustrating the linkages between genes and enriched GO terms within the Biological Process (BP) category. Each node represents a GO term, and lines (edges) indicate shared genes enriched in multiple GO terms, forming interconnections between the terms and genes. (B) Network showing the linkages between genes and enriched GO terms within the Molecular Function (MF) category. (C) Network of genes and enriched GO terms within the Cellular Component (CC) category. (D) Stage-specific expression analysis of genes enriched in GO biological processes. The underlined transcript IDs were selected for the qRT-PCR validation experiment.

Figure 8

qRT-PCR validation and paraffin sections of petals and sepals in Den. Sect. Spatulata hybrids. (A) qRT-PCR validation of six selected genes across developmental Stages 1-4 in petals and sepals of Den. Sect. Spatulata hybrids. Statistical significance, determined by Student’s t-test, is indicated as follows: *P < 0.05; **P < 0.001. Number of replicates = 3. (B) Paraffin sections of petals and sepals from Den. Sect. Spatulata hybrids. Stages 1 to 4 display paraffin sections of petals and sepals from H5 at different developmental stages, while H2 represents sections of petals and sepals at the full-bloom stage. Red arrows above and below the sepals denote the key differences in cell number and size observed in this study.