Heading Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a significant dietary vegetable for its edible heading leaves in Asia countries. The new purple anthocyanin-rich pure line (11S91) was successfully bred, and the anthocyanins were mainly distributed in 2-3 cell layers beneath the leaf epidermis, whereas siliques and stems accumulated only a cell layer of anthocyanins. The anthocyanins of 11S91 were more stable at pHs below 3.0 and temperatures below 45 °C. The total antioxidant ability was highly positive correlated with the anthocyanin content in 11S91. Thirty-two anthocyanins were separated and identified, and 70% of them were glycosylated and acylated cyanidins. The four major anthocyanins present were cyanidin-3-sophoroside(p-coumaroyl)-5-glucoside(malonyl), cyanidin-3-sophoroside(ferulyl)-5-glucoside(malonyl), cyanidin-3-sophoroside(sinapyl-p-coumaroyl)-5-glucoside(malonyl), and cyanidin-3-sophoroside-(sinapyl-ferulyl)-5-glucoside(malonyl). According to the expression of biosynthetic genes and the component profile of anthocyanins in 11S91 and its parents, regulatory genes BrMYB2 and BrTT8 probably activate the anthocyanin biosynthesis but other factors may govern the primary anthocyanins and the distribution.
Anthocyanins are important secondary metabolites in plants, but information on anthocyanin biosynthesis mechanisms in Chinese cabbage is limited. The new purple head Chinese cabbage cultivar 11S91 was analyzed, and an R2R3-MYB regulatory gene BrMYB2, located on chromosome A07, controlling the dominant purple-head trait was isolated. High expression of BrMYB2 generated a large accumulation of anthocyanins in 11S91, accompanied by highly upregulated BrTT8, BrF3′H, BrDFR1, BrANS1, BrUGTs, BrATs, and BrGSTs. 11S91 inherited the purple locus from purple trait donor 95T2-5, and they shared consensus CDSs and gDNAs with those of BrMYB2 (cBrMYB2 and gBrMYB2). Two SNPs in cBrMYB2 in 11S91 did not cause loss of function; in addition to several SNPs at both ends of intron 1, a large deletion had occurred in intron 1 of gBrMYB2 in 11S91. Genetic transformation of Arabidopsis showed that gBrMYB2 overexpression lines presented deeper purple color and higher expression than did the cBrMYB2 and cBrmyb2 lines, whereas gBrmyb2 with a long intron 1 did not cause the purple phenotype. We first show that BrMYB2 promotes anthocyanin biosynthesis under the control of the short intron 1 of gBrMYB2 in purple head Chinese cabbage, and gBrmyb2 with a long intron 1 represses anthocyanin production in white head Chinese cabbage. This evidence provides a new understanding of anthocyanin biosynthesis and purple germplasm generation in Brassica vegetables.
To elucidate the effect of low temperature on anthocyanin biosynthesis in purple head Chinese cabbage, we analyzed anthocyanin accumulation and related gene expression in the seedlings of purple head Chinese cabbage, white head parent Chinese cabbage, and its purple male parent under a normal 25 °C temperature and a low 12 °C temperature. Anthocyanin accumulation in purple lines was strongly induced by low temperature, and the total anthocyanin content of seedlings was significantly enhanced. In addition, nearly all phenylpropanoid metabolic pathway genes (PMPGs) were down-regulated, some early biosynthesis genes (EBGs) were up-regulated, and nearly all late biosynthesis genes (LBGs) directly involved in anthocyanin biosynthesis showed higher expression levels in purple lines after low-temperature induction. Interestingly, a R2R3-MYB transcription factor (TF) gene ‘BrMYB2’ and a basic-helix-loop-helix (bHLH) regulatory gene ‘BrTT8’ were highly up-regulated in purple lines after low temperature induction, and two negative regulatory genes ‘BrMYBL2.1’ and ‘BrLBD38.2’ were up-regulated in the white line. BrMYB2 and BrTT8 may play important roles in co-activating the anthocyanin structural genes in purple head Chinese cabbage after low-temperature induction, whereas down-regulation of BrMYB2 and up-regulation of some negative regulators might be responsible for white head phenotype formation. Data presented here provide new understanding into the anthocyanin biosynthesis mechanism during low temperature exposure in Brassica crops.
The proanthocyanidin (PA) is the main flavonoids which affect the seed coat color in Brassica species. In this paper, characteristics of color development and accumulation of flavonoids were analyzed in the seeds of brown-seeded (B147) and yellow-seeded (B80) heading Chinese cabbage (Brassica rapa L. ssp. Pekinensis). It is found that the content of phenolic compounds in B147 were significantly more than that of B80 by using dimethylaminocinnamaldehyde (DMACA) staining and toluidine blue O (TBO) staining. In previous studies, the locus associated with seed coat color has been mapped. The results of whole genome re-sequencing showed that there are large fragment deletions variation in the mapping region between the brown-seeded parent ‘92S105’ and the yellow-seeded parent ‘91-125.’ Based on the B. rapa genome annotation information, the TRANSPARENT TESTA GLABRA 1 (TTG1), is likely to be the candidate gene controlling seed coat color. A 94-base deletion was found in the 96th base downstream of the initiation codon in the TTG1 of yellow seed, thus, the termination codon TGA was occurred in the 297th base which makes the full length of TTG1 of yellow seed is 300 bp. Based on the differential sequences of TTG1 of brown and yellow seed, a functional marker, Brsc-yettg1, was developed to detect the variation of TTG1. Quantitative real-time PCR analysis of BrTTG1 in different tissues showed that expression levels of BrTTG1 was not tissue-specific. During the whole seed development period, the expression of BrTTG1 in B147 was higher than that of B80. The expression levels of four structural genes, BrDFR, BrANS, BrANR1, and BrANR2 in B147 were also higher than those in B80. The co-segregation molecular markers obtained in this report and TTG1 related information provide a basis for further understanding of the molecular mechanism of seed coat color in heading Chinese cabbage.
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