Global dissection of alternative splicing uncovers transcriptional diversity in tissues and associates with the flavonoid pathway in tea plant (Camellia sinensis)

Abstract

Background: Alternative splicing (AS) regulates mRNA at the post-transcriptional level to change gene function in organisms. However, little is known about the AS and its roles in tea plant (Camellia sinensis), widely cultivated for making a popular beverage tea.

Results: In our study, the AS landscape and dynamics were characterized in eight tissues (bud, young leaf, summer mature leaf, winter old leaf, stem, root, flower, fruit) of tea plant by Illumina RNA-Seq and confirmed by Iso-Seq. The most abundant AS (~ 20%) was intron retention and involved in RNA processes. The some alternative splicings were found to be tissue specific in stem and root etc. Thirteen co-expressed modules of AS transcripts were identified, which revealed a similar pattern between the bud and young leaves as well as a distinct pattern between seasons. AS events of structural genes including anthocyanidin reductase and MYB transcription factors were involved in biosynthesis of flavonoid, especially in vegetative tissues. The AS isoforms rather than the full-length ones were the major transcripts involved in flavonoid synthesis pathway, and is positively correlated with the catechins content conferring the tea taste. We propose that the AS is an important functional mechanism in regulating flavonoid metabolites.

Conclusion: Our study provides the insight into the AS events underlying tea plant's uniquely different developmental process and highlights the important contribution and efficacy of alternative splicing regulatory function to biosynthesis of flavonoids.

Keywords: Alternative splicing; Camellia sinensis; Flavonoid; Tissue-specificity.

Fig. 1

Comparative analysis of different AS isoforms among eight tissues of tea plant. a Venn diagram showing the common and unique AS genes determined with Illumina and SMRT sequencing. b Distribution of different types of alternative splicing events in eight tissues. c Overlap of AS genes among eight tissues. d KEGG pathway enrichment for AS genes from eight tissues. The area of each colored circle is proportional to the size scale of genes involved in each pathway. The color indicates the q-value, and the x-axis is the Rich Factor

Fig. 2

Co-expression network analysis. WGCNA analysis of AS genes in eight tissues. Each column corresponds to a specific tissue (B, bud; YL, young leaf; SML, summer mature leaf; WOL, winter old leaf; S, stem; R, root; FL, flower; FR, fruit). Each row corresponds to a module. The number of genes in each module is indicated on the left box. The color and number of each cell at the row-column intersection indicates the correlation coefficient between the module and the tissue type. A high degree of correlation between a specific module and the tissue type is indicated by dark red

Fig. 3

Characteristic analysis of tissue-specific AS genes. a Distribution of different types of alternative splicing events in eight tissues. b Heat map showing the transcript level in FPKM of each tissue-specific AS gene from the pink (stem) module, green (root) module, red (flower) module and brown (fruit), respectively. Red and blue colors represent high and low levels of transcript abundance, respectively

Fig. 4

Alternatively spliced isoforms of flavonoid-related genes in tea plant. The red asterisk indicates the position of PTC. The full-length and AS isoforms on the gel images are denoted with red and black triangles, respectively. The orange solid dot indicates the targeted AS transcripts were identified in specific tissues (B, bud; YL, young leaf; SML, summer mature leaf; WOL, winter old leaf; S, stem; R, root; FL, flower; FR, fruit)

Fig. 5

Expression patterns of flavonoid-related AS transcripts. a Heat map showing the FPKM of non-AS and AS transcripts in eight tissues (B, bud; YL, young leaf; SML, summer mature leaf; WOL, winter old leaf; S, stem; R, root; FL, flower; FR, fruit). Red and blue colors represent high and low transcript abundance, respectively. b Read coverage graphs of TEA003146.1 and TEA012130.1 derived from eight tissues viewed in IGV and validated by semi-quantitative RT-PCR. The red dotted boxes indicate the positions of alternative splicing. The RT-PCR bands are shown in the gel images beneath the graphs. The CsGAPDH gene was used as the internal control

Fig. 6

Hypothetical model for functions of alternative splicing in tea plant. The biosynthesis of flavonoid is active in vegetative tissues (bud, young leaf, summer mature leaf and winter old leaf) but passive in other tissues (stem, root, flower and fruit). As details of the processes of flavonoid biosynthesis, the full-length and AS transcripts synergistically functions and positive and negative relationship are exhibited between them at transcription level