Red Chinese Cabbage Transcriptome Analysis Reveals Structural Genes and Multiple Transcription Factors Regulating Reddish Purple Color

Abstract

Reddish purple Chinese cabbage (RPCC) is a popular variety of Brassica rapa (AA = 20). It is rich in anthocyanins, which have many health benefits. We detected novel anthocyanins including cyanidin 3-(feruloyl) diglucoside-5-(malonoyl) glucoside and pelargonidin 3-(caffeoyl) diglucoside-5-(malonoyl) glucoside in RPCC. Analyses of transcriptome data revealed 32,395 genes including 3345 differentially expressed genes (DEGs) between 3-week-old RPCC and green Chinese cabbage (GCC). The DEGs included 218 transcription factor (TF) genes and some functionally uncharacterized genes. Sixty DEGs identified from the transcriptome data were analyzed in 3-, 6- and 9-week old seedlings by RT-qPCR, and 35 of them had higher transcript levels in RPCC than in GCC. We detected cis-regulatory motifs of MYB, bHLH, WRKY, bZIP and AP2/ERF TFs in anthocyanin biosynthetic gene promoters. A network analysis revealed that MYB75, MYB90, and MYBL2 strongly interact with anthocyanin biosynthetic genes. Our results show that the late biosynthesis genes BrDFR, BrLDOX, BrUF3GT, BrUGT75c1-1, Br5MAT, BrAT-1, BrAT-2, BrTT19-1, and BrTT19-2 and the regulatory MYB genes BrMYB90, BrMYB75, and BrMYBL2-1 are highly expressed in RPCC, indicative of their important roles in anthocyanin biosynthesis, modification, and accumulation. Finally, we propose a model anthocyanin biosynthesis pathway that includes the unique anthocyanin pigments and genes specific to RPCC.

Dynamic Changes of the Anthocyanin Biosynthesis Mechanism During the Development of Heading Chinese Cabbage (Brassica rapa L.) and Arabidopsis Under the Control of BrMYB2

“…The scale bar is 4.5 cm. purple Chinese cabbage, interspecific-crossing between a green Chinese cabbage and a red cabbage was used, combined with recurrent backcrossing to acquire red aneuploid individuals of Chinese cabbage (Lee et al, 2018), for which a series of upregulated ABGs were reported (Rameneni et al, 2020). Unlike those breeding approaches, we relied on the intersubspecificcrossing between a white head Chinese cabbage and a purple flowering Chinese cabbage, with the control of the key BrMYB2 gene located on chromosome A07 (He et al, 2016(He et al, , 2020b.…”

“…The red or purple germplasm of Chinese cabbage has been well discussed when adopting different conceptions of breeding via hybridization ( Zhang et al, 2016 ; Lee et al, 2018 ; He et al, 2020b ). Among them, Zhang et al (2016) created a purple head Chinese cabbage by crossing a common Chinese cabbage with a red color B. juncea using the embryo rescue technique and continuous backcrossing, in which the purple genes were translocated onto chromosome A02; in another breeding case of purple Chinese cabbage, interspecific-crossing between a green Chinese cabbage and a red cabbage was used, combined with recurrent backcrossing to acquire red aneuploid individuals of Chinese cabbage ( Lee et al, 2018 ), for which a series of upregulated ABGs were reported ( Rameneni et al, 2020 ). Unlike those breeding approaches, we relied on the intersubspecific-crossing between a white head Chinese cabbage and a purple flowering Chinese cabbage, with the control of the key BrMYB2 gene located on chromosome A07 ( He et al, 2016 , 2020b ).…”

“…Work with Solanaceae species such as chili pepper showed that CHS , CHI , F3H , ANS , and ANP are downregulated at some developmental stages of an anthocyanin non-accumulator type, whereas these genes together with ANS , DFR , and GST are developmentally upregulated in plant phenotypes with different contents of anthocyanin accumulation ( Aza-González et al, 2013 ). In addition, reports on Brassica crops such as red cabbage have investigated their anthocyanin accumulation and gene expression in response to light and dark conditions during seedlings’ development; more recent work has investigated the ABGs’ expression patterns of purple or red Brassica crops in some key stages of the plant development (i.e., seedlings, heads, or both), by using transcriptome sequencing techniques, to effectively reveal the potential mechanism of anthocyanin biosynthesis ( Mushtaq et al, 2016 ; Xie et al, 2016 ; Zhang et al, 2017 ; Jeon et al, 2018 ; Rameneni et al, 2020 ). Based on these analyses, we find that these studies primarily select a special developing period and a series of typical ABGs involved downstream or upstream of the anthocyanin biosynthesis pathway, the comparisons between the different developing periods, homologous copies, and tissues of plants combining with close genetic backgrounds were rarely studied.…”

“…capitata f. rubra , 2n = CC = 18), then combined with a colchicine treatment to form allotetraploids and a recurrent backcrossing, to acquire the red aneuploid individuals of Chinese cabbage ( Lee et al, 2018 ). The new reddish-purple Chinese cabbage appeared as a red head phenotype with introduced dominant genes and a retarded growth character, whose transcriptome analysis showed the LBGs BrDFR , BrLDOX , BrUF3GT , BrUGT75c1-1 , Br5MAT , BrAT-1 , BrAT-2 , BrTT19-1 , and BrTT19-2 and the regulatory MYB genes BrMYB90 , BrMYB75 , and BrMYBL2-1 were highly expressed in it ( Lee et al, 2018 ; Rameneni et al, 2020 ). Unlike those distant hybridization forms of breeding, we have created a novel, purple heading Chinese cabbage based on the inspiration of intersubspecific hybridization, for which a white heading Chinese cabbage (94S17, 2n = AA = 20) and a purple flowering Chinese cabbage (95T2-5, 2n = AA = 20) are used in the hybridization ( He et al, 2016 ; Wu et al, 2016 ).…”

“…Additionally, four ANS s and 12 DFR s of B. rapa were investigated under cold and freezing conditions: BrANS2 , BrDFR1 , BrDFR3 , BrDFR5 , BrDFR6 , and BrDFR10 genes all responded to cold and freezing stress treatments, whereas BrANS1 , BrANS3 , BrDFR2 , BrDFR4 , BrDFR8 , and BrDFR 9 only highly responded to cold stress in the purple-pigmented B. rapa plants ( Ahmed et al, 2014 , 2015 ). In the reddish purple head Chinese cabbage, results indicate that BrPAL , BrPAL2 , BrPAL4 , BrC4H , Br4CL2 , BrCHS , BrCHI , BrCHI1 , BrF3H , and BrF3′H-1 may be involved in the early phase of anthocyanin biosynthesis; however, LBGs BrDFR , BrLDOX , BrUF3GT , BrUGT75c1-1 , Br5MAT , BrAT-1 , BrAT-2 , BrTT19-1 , and BrTT19-2 and the regulatory MYB genes BrMYB90 , BrMYB75 , and BrMYBL2-1 might play important roles in the anthocyanin biosynthesis in their purple plants ( Rameneni et al, 2020 ). Hence, that evidence raises the pressing questions of whether all ABGs are involved in the purple trait formation and how do they operate during the development of the purple heading Chinese cabbage.…”