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. 2025 Jul 11:16:1627126.
doi: 10.3389/fpls.2025.1627126. eCollection 2025.

Comprehensive analysis of alternative splicing in Rosa roxburghii Tratt reveals its role in flavonoid synthesis

Yanlin An  1 , Jiahua Wu  1 , Yani Chen  1 , Shize Li  1   2
Affiliations

Comprehensive analysis of alternative splicing in Rosa roxburghii Tratt reveals its role in flavonoid synthesis

Yanlin An et al. Front Plant Sci. .

Abstract

Introduction: Alternative splicing (AS) plays an important role in the synthesis of plant metabolites. Chestnut rose is a fruit with rich metabolites and health benefits. However, the role of AS in its metabolite synthesis is rarely reported.

Methods: The transcriptome data of eight tissues of Rosa roxburghii were analyzed by using Trimmomatic, Hisat2 and StringTie software. AStalavista tool was used to identify alternative splicing (AS) events, which were further analyzed with IGV browser and other tools. The WGCNA package in R software was applied to construct co-expression networks. AS events were validated by RT-qPCR, and the RrActin was used as an internal control to analyze the transcription expression pattern.

Results: In this study, the AS landscape were characterized in different tissues of chestnut rose. The data showed that 8586 genes could undergo AS and a total of 49,523 AS events were generated. Among them, tissue-specific AS genes were found in leaves, flowers and fruits. The content of flavonoids in the samples was detected and WGCNA analysis was performed with the AS genes. Five key modules of AS genes related to flavonoid synthesis were identified, and 4CL, ANR, DFR, MYB and other AS genes were validated by PCR and sequencing. In addition, qRT-PCR analysis revealed that the expression level of basic helix-loop-helix (bHLH) transcription factor AS transcript was higher than its full-length transcript, and it was highly expressed in FR1 and significantly correlated with flavonoids.

Discussion: Our research identified AS events in different tissues of chestnut rose and revealed their important functions in flavonoid synthesis. This study provides the basis for the molecular mechanism of flavonoids in chestnut rose.

Keywords: Rosa roxburghii Tratt; WGCNA; alternative splicing; flavonoid synthesis; molecular mechanism.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Analysis of AS genes in chestnut rose. (A) Statistics on the number of AS genes of four main types in eight samples. (B) Up-set venn diagram displays specific and conserved AS genes in eight samples. (C) KEGG enrichment analysis of differentially expressed AS genes with P value < 0.01. The color and size of the circles represent the p-values and gene number, respectively.
Figure 2
Figure 2
Analysis of differential flavonoid content in eight samples. Metabolite levels were clustered based on their average content. The blue and red colors in the heatmap represent low and high material content, respectively.
Figure 3
Figure 3
WGCNA of differentially expressed AS genes. The color blocks on the left represent the clustering of AS genes. The blue and red colors in the figure represent the negative and positive correlations between gene modules and metabolite content.
Figure 4
Figure 4
Flavonoid biosynthetic pathway and AS gene expression analysis. The red dashed box represents the differential flavonoid substances identified in the study. Heat map showing the relative expression level of AS genes with mean values of three biological replicates in eight samples.
Figure 5
Figure 5
AS isoforms associated with flavonoid content. Agarose gel electrophoresis is shown on the left and green triangles represent AS and annotated transcripts respectively. The red dashed box represents the position where AS event occurs.
Figure 6
Figure 6
Expression of AS genes related to flavonoid content analyzed by qRT-PCR. 2-△Ct is used to represent its relative expression level. The lowercase letters represent significant differences at the P < 0.05 level.
See this image and copyright information in PMC

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