Transcriptomic dynamics changes related to anthocyanin accumulation in the fleshy roots of carmine radish (Raphanus sativus L.) characterized using RNA-Seq

Abstract

Carmine radish is famous for containing a natural red pigment (red radish pigment). However, the expression of anthocyanin biosynthesis-related genes during the dynamic development stages of the fleshy roots in carmine radish has not been fully investigated. Here, based on HPLC quantification of anthocyanin levels from our previous study, young fleshy roots of the carmine radish "Hongxin 1" obtained at the dynamic development stages of fleshy roots (seedling stage (SS), initial expansion (IE), full expansion (FE), bolting stage (BS), initial flowering stage (IFS), full bloom stage (FBS) and podding stage (PS)) were used for RNA-Seq. Approximately 126 comodulated DEGs related to anthocyanin biosynthesis (common DEGs in the dynamic growth stages of fleshy roots in carmine radish) were identified, from which most DEGs appeared to be likely to participate in anthocyanin biosynthesis, including two transcription factors, RsMYB and RsRZFP. In addition, some related proteins, e.g., RsCHS, RsDFR, RsANS, RsF'3H, RsF3GGT1, Rs3AT1, RsGSTF12, RsUFGT78D2 and RsUDGT-75C1, were found as candidate contributors to the regulatory mechanism of anthocyanin synthesis in the fleshy roots of carmine radish. In addition, 11 putative DEGs related to anthocyanin synthesis were evaluated by qRT-PCR via the (2-ΔΔCT) method; the Pearson correlation analysis indicated excellent concordance between the RNA-Seq and qRT-PCR results. Furthermore, GO enrichment analysis showed that "anthocyanin-containing compound biosynthetic process" and "anthocyanin-containing compound metabolic process" were commonly overrepresented in the dynamic growth stages of fleshy roots after the initial expansion stage. Moreover, five significantly enriched pathways were identified among the DEGs in the dynamic growth stages of fleshy roots in carmine radish, namely, flavonoid biosynthesis, flavone and flavonol biosynthesis, diterpenoid biosynthesis, anthocyanin biosynthesis, and benzoxazinoid biosynthesis. In conclusion, these results will expand our understanding of the complex molecular mechanisms of anthocyanin biosynthesis in the fleshy roots of carmine radish and the putative candidate genes involved in this process.

Keywords: Anthocyanin biosynthesis; Anthocyanins; Differential expression genes (DEGs); KEGG pathway enrichment; Radish (Raphanus sativus L.).

Figure 1. Soft clusters of normalized expression levels for all global expressed genes involved in the dynamics growing stages of fleshy roots in carmine radish.

A total of nine clusters (A-I) with the different level of expression changes were identified. Horizontal axis represents growing stages of fleshy roots (“SS_root”, “IE_root”, “FE_root”, “BS_root”, “IFS_root”, “FBS_root” and “PS_root”). The vertical axis represents expression changes.

Figure 2. Transcriptional changes of DEGs involved in the dynamics growing stages of fleshy roots in carmine radish.

(A) Statistic of differentially expression genes (including up-regulated and down-regulated in each comparison groups) in the dynamics growing stages of fleshy roots (“IE_root”, “FE_root”, “BS_root”, “IFS_root”, “FBS_root” and “PS_root”), compared with “SS_root” group. (B) Venny graph of co-modulated DEGs (Common DEGs in the dynamic growing stages of fleshyroot in carmine radish). (C) Clustering and heat map of common differentially expressed (Co-modulated genes) based on the expression profiles in the dynamics growing stages of fleshy roots (“IE_root”, “FE_root”, “BS_root”, “IFS_root”, “FBS_root” and “PS_root”), compared with “SS_root” group.

Figure 3. Functional enrichment analysis of differentially expressed genes (DEGs) related to the dynamics growing stages of fleshy roots in carmine radish.

(A) Enriched GO terms of DEGs related to the dynamics growing stages of fleshy roots in radish. GO terms are plotted on the ordinate, and the enrichment factor (rich factor) is plotted on the abscissa. The color of points represents the q-value, and the size of points represents the number of DEGs mapped to the reference pathway. Legends for the color scale of q-values and size-scaling of the number of DEGs are shown to the right of the plot. (B) Pathway enrichment analysis among differentially expressed genes related to anthocyanin synthesis in radish. Enriched KEGG pathway terms divided by the dynamics growing stages (“IE_root”, “FE_root”, “BS_root”, “IFS_root”, “FBS_root” and “PS_root”), compared with “SS_root”. Red color indicates statically overrepresented.

Figure 4. (A–K) Transcriptional analysis of anthocyanin synthesis-related genes (ASRGs) identified in fleshy roots obtained from the dynamics development stage of fleshy roots (seedling stage (SS), initial expansion (IE), full-expansion (FE), bolting stage (BS), initial.

flowering stage (IFS); full-bloom stage (FBS) and podding stage (PS)) in carmine radish “Hongxin 1” using qRT-PCR. Relative gene expression levels were normalized against actin transcript levels, and log2 scale for fold change of gene expression in the development stage of fleshy roots comprising of “SS”, “IE”, “FE”, “BS”, “IFS”, “FBS” and “PS” was shown. The standard error calculated from three biological replicates and significant (P < 0.05) difference identified by uncorrected Fisher’s LSD test in multiple comparisons after two-way ANOVA are indicated by error bars and stars, respectively.

Figure 5. Correlations of transcript levels of candidate DEGs related to anthocyanin synthesis traits in carmine radish between RNA-seq and qPCR data.

Validation of candidate Co-modulated DEGs involved in the dynamics growing stages of fleshy roots in carmine radish using qRT-PCR and then correlation between RNA-seq and qPCR data were conducted. Each RNA-seq expression data was plotted against that from quantitative real-time PCR and fit into a linear regression. Both x- and y-axes were shown in log2 scale and each color represented a different gene.