Integrated transcriptomic and WGCNA analyses reveal candidate genes regulating mainly flavonoid biosynthesis in Litsea coreana var. sinensis

Abstract

Litsea coreana Levl. var. sinensis (Allen) Yang et P. H. Huang is a popular ethnic herb and beverage plant known for its high flavonoid content, which has been linked to a variety of pharmacological benefits and crucial health-promoting impacts in humans. The progress in understanding the molecular mechanisms of flavonoid accumulation in this plant has been hindered due to the deficiency of genomic and transcriptomic resources. We utilized a combination of Illumina and Oxford Nanopore Technology (ONT) sequencing to generate a de novo hybrid transcriptome assembly. In total, 126,977 unigenes were characterized, out of which 107,977 were successfully annotated in seven public databases. Within the annotated unigenes, 3,781 were categorized into 58 transcription factor families. Furthermore, we investigated the presence of four valuable flavonoids-quercetin-3-O-β-D-galactoside, quercetin-3-O-β-D-glucoside, kaempferol-3-O-β-D-galactoside, and kaempferol-3-O-β-D-glucoside in 98 samples, using high-performance liquid chromatography. A weighted gene co-expression network analysis identified two co-expression modules, MEpink and MEturquoise, that showed strong positive correlation with flavonoid content. Within these modules, four transcription factor genes (R2R3-MYB, NAC, WD40, and ARF) and four key enzyme-encoding genes (CHI, F3H, PAL, and C4H) emerged as potential hub genes. Among them, the R2R3-MYB (LcsMYB123) as a homologous gene to AtMYB123/TT2, was speculated to play a significant role in flavonol biosynthesis based on phylogenetic analysis. Our findings provided a theoretical foundation for further research into the molecular mechanisms of flavonoid biosynthesis. Additionally, The hybrid transcriptome sequences will serve as a valuable molecular resource for the transcriptional annotation of L. coreana var. sinensis, which will contribute to the improvement of high-flavonoid materials.

Keywords: De novo transcriptome sequencing; Litsea coreana var. sinensis; Candidate genes; Flavonoids; WGCNA.

Fig. 1

The main flavonoid content analysis of L. coreana var. sinensis. (A) Flavonoid contents assessed by HPLC fingerprint analysis (mg/g dry weight). Different letters indicate significant differences (P < 0.05). (B) Typical HPLC chromatograms of L. coreana var. sinensis

Fig. 2

Gene annotation and functional classification. (A) Homologous species distribution annotated in the NR database. (B) The KOG categories of the unigenes. (C) Annotation in KEGG pathway classification. The image was created by the author, and KEGG copyright permission was obtained

Fig. 3

The results of WGCNA. (A) Correlation coefficient between flavonoids and module eigengenes with red and blue representing positive and negative correlations, respectively. Each column corresponds to K-3-gal, Q-3-glu, Q-3-gal and K-3-glu values, and each row represents a module. p-values are shown in parentheses. The x-axis represents the four flavonoids, while the y-axis represents various modules. (B) The number of genes in each module. (C) The number of TFs in each module

Fig. 4

Bubble maps of top 20 KEGG-enriched pathways of genes in pink and turquoise modules. (A) pink module. (B) turquoise module. The x-axis and y-axis represent the rich factor and pathway name, respectively. The ‘Rich Factor’ refers to the ratio of genes in the target gene set that belong to a specific pathway, compared to all the genes present in that pathway within the background gene set. The image was created by the author, and KEGG copyright permission was obtained

Fig. 5

Hub gene visualization of the pink and turquoise modules. (A) pink module. (B) turquoise module. The larger the circle, the darker the color, and the greater the degree value

Fig. 6

Expression patterns of genes involved in flavonoid biosynthesis pathways in pink and turquoise modules. (A) Structural genes expression levels. (B) TFs genes expression levels. The color scale from green to red represents the TPM values from low to high. The hub genes identified by WGCNA were represented in the round rectangular box. DZ5, DZ7, DZ15: the three samples with the lowest total amount of K-3-gal, Q-3-glu, Q-3-gal and K-3-glu. MT15, MT11_1, XS7: the three samples with the highest total amount of K-3-gal, Q-3-glu, Q-3-gal and K-3-glu

Fig. 7

Pathway view heatmap of flavonoids and hub gene expressions in different L. coreana var. sinensis samples. Each sample is represented by a column, and each flavonoid/gene is represented by a row. Each flavonoid/gene expression profile is represented by a unique color, which accurately represents its relative abundance. Red indicates high-abundant flavonoids, whereas low-abundant flavonoids are presented in green. Red indicates high gene expression levels

Fig. 8

Phylogenetic analysis and protein sequence alignment of LcsMYB123. (A) Phylogenetic analysis of flavonoid-regulating MYBs in different plants. (B) Multi-alignment of the protein sequence of MYBs from different species. The R2 and R3 domains were underlined

Fig. 9

qRT-PCR verification. (A) Expression of 10 genes related to flavonoid biosynthesis. (B) Pearson’s correlation of gene expression between RNA-seq and qRT-PCR. The columns show RNA-seq expression (TPM) on the left y-axis, while lines represent qRT-PCR relative expression (2 ^−ΔΔCq) on the right-axis