Genetic and Comparative Transcriptome Analysis Revealed DEGs Involved in the Purple Leaf Formation in Brassica juncea

Abstract

Brassica juncea is an important dietary vegetable cultivated and consumed in China for its edible stalks and leaves. The purple leaf mustard, which is rich in anthocyanins, is eye-catching and delivers valuable nutrition. However, the molecular mechanism involved in anthocyanin biosynthesis has not been well studied in B. juncea. Here, histological and transcriptome analyses were used to characterize the purple leaf color and gene expression profiles. Free-hand section analysis showed that the anthocyanin was mainly accumulated in the adaxial epidermal leaf cells. The anthocyanin content in the purple leaves was significantly higher than that in the green leaves. To investigate the critical genes and pathways involved in anthocyanin biosynthesis and accumulation, the transcriptome analysis was used to identify the differentially expressed genes (DEGs) between the purple and green leaves from the backcrossed BC3 segregation population in B. juncea. A total of 2,286 different expressed genes were identified between the purple and green leaves. Among them, 1,593 DEGs were up-regulated and 693 DEGs were down-regulated. There were 213 differently expressed transcription factors among them. The MYB and bHLH transcription factors, which may regulate anthocyanin biosynthesis, were up-regulated in the purple leaves. Interestingly, most of the genes involved in plant-pathogen interaction pathway were also up-regulated in the purple leaves. The late biosynthetic genes involved in anthocyanin biosynthesis were highly up-regulated in the purple leaves of B. juncea. The up regulation of BjTT8 and BjMYC2 and anthocyanin biosynthetic genes (BjC4H, BjDFR, and BjANS) may activate the purple leaf formation in B. juncea. This study may help to understand the transcriptional regulation of anthocyanin biosynthesis in B. juncea.

Keywords: Brassica juncea; anthocyanin; gene expression; purple leaves; transcriptome.

FIGURE 1

Morphology of purple leaf and anthocyanin content in B. juncea. (A) Upper epidermis of green (LvYi) and purple (ZiYi) leaf. (B) Lower epidermis of green and purple leaf. (C) Transverse section of a green leaf. Bars = 200 μm. (D) Transverse section of a purple leaf. Bars = 200 μm. (E) Total anthocyanin contents of green and purple leaves. **Signifcantly diferent at P < 0.01.

FIGURE 2

Different expressed genes between purple and green leaf. (A) The volcano map of DEGs between ZiYi and LvYi. Each dot represents a gene. (B) The number of up- and down-regulated genes in ZiYi. (C) KEGG metabolism pathway analysis of the DEGs between ZiYi and LvYi. Histograms represent the metabolism pathway distribution; the X axial represents the number of genes that have been annotated to the metabolism pathway.

FIGURE 3

Distinct expression patterns of transcription factors. (A) The number of different expressed transcription factors between ZiYi and LvYi. (B) Gene numbers of different type transcription factors among the DEGs between ZiYi and LvYi. (C) Heatmaps representing the distinct expression patterns of transcription factors between ZiYi and LvYi. BjP1, BjP2, and BjP3 representative three biological replication in ZiYi. BjG1, BjG2, and BjG3 representative three biological replication in LvYi. (D) Comparison of the clusters of TF families.

FIGURE 4

Diagram and heatmaps of genes involved the anthocyanin biosynthesis pathway. (A) Schematic diagram of genes involved in the anthocyanin biosynthesis pathway. (B) Heatmaps of DEGs involved in the accumulation of anthocyanin in ZiYi B. juncea.

FIGURE 5

qRT-PCR analysis the expression pattern of genes involved in anthocyanin biosynthesis B. juncea. The purple color represents transcript expression level of genes in ZiYi B. juncea with purple leaf. The green color represents transcript expression level of genes in LvYi B. juncea with green leaf. *Signifcantly diferent at P < 0.05; **Signifcantly diferent at P < 0.01.