Abstract
Main conclusion
BraANS.A3 was the key gene controlling purple leaf color in pak choi, and two short fragments of promoter region in green pak choi might be interfering its normal expression.
Abstract
Pak choi (B. rapa L. ssp. chinensis) is an influential and important vegetable with green, yellow, or purple leaves that is cultivated worldwide. The purple leaves are rich in anthocyanins, but the underlying genetics and evolution have yet to be extensively studied. Free-hand sections of the purple leaves indicated that anthocyanins mainly accumulate throughout the adaxial and abaxial epidermal leaf cells. Segregation analyses of an F2 population of a B. rapa ssp. chinensis L. purple leaf mutant ZBC indicated that the purple trait is controlled by an incompletely dominant nuclear gene. Bulked segregant analysis (BSA) showed that the key genes controlling the trait were between 24.25 and 38.10 Mb on chromosome A03 of B. rapa. From the annotated genes, only BraA03g050560.3C, homologous to Arabidopsis AtANS, was related to the anthocyanin synthesis pathway. Genome annotation results and transcriptional sequencing analyses revealed that the BraANS.A3 gene was involved in the purple leaf trait. qRT-PCR analyses showed that BraANS.A3 was highly upregulated in ZBC but hardly expressed in the leaves of an inbred homozygous line of B. campestris ssp. chinensis L. green leaf mutant WTC, indicating that BraANS.A3 played a key role catalyzing anthocyanin synthesis in ZBC. Full-length sequence alignment of BraANS.A3 in WTC and ZBC showed that it was highly conserved in the gene region, with significant variation in the promoter region. In particular, the insertion of two short fragments of the promoter region in WTC may interfere with its normal expression. The promoter regions of ANS in six Brassica species all had multiple cis-elements involved in responses to abscisic acid, light, and stress, suggesting that ANS may be involved in multiple metabolic pathways or biological processes. Protein–protein interactions predicted that BraANS.A3 interacts with virtually all catalytic proteins in the anthocyanin synthesis pathway and has a strong relationship with Transparent Testa 8 (TT8). These results suggest that BraANS.A3 promotes anthocyanin accumulation in purple pak choi and provide new insights into the functional analysis of anthocyanin-related genes in Chinese cabbage and transcriptional regulatory networks.
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Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA881503.
Abbreviations
- ANS:
-
Anthocyanidin synthase
- AT:
-
Acyltransferase
- BSA:
-
Bulked segregant analysis
- C4H:
-
Cinnamate-4-hydroxylase
- CHS:
-
Chalcone synthase
- CHI:
-
Chalcone isomerase
- DEG:
-
Differentially expressed gene
- DFR:
-
Dihydroflavonol 4-reductase
- FLS:
-
Flavonol synthase
- LBD:
-
LATERAL ORGAN BOUNDARIES (LOB) DOMAIN
- MYBL2:
-
Arabidopsis MYB-like 2
- TT8:
-
Transparent Testa 8
- WTC:
-
B. campestris Ssp. chinensis L. green leaf mutant
- ZBC:
-
B. rapa Ssp. chinensis L. purple leaf mutant
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Funding
This research was funded by the National Natural Science Foundation of China, under Grant Nos. 32160454 and 32260469; the Natural Science Foundation of Jiangxi Province, under Grant No. 20212BAB215002; the Open Fund Project of Jiangxi Key Laboratory of Oil Crop Biology, under Grant No. YLKFKT202202.
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425_2023_4171_MOESM1_ESM.png
Supplementary file1 Supplementary Fig. S1 Genetic analysis of testa color in purple pakchoi and the overview of this study. a Phenotypic and genetic analysis of ZBC and WTC crosses. b Anthocyanin content of ZBC and WTC leaves. Error bars indicate SE from three replicates (**P<0.01). c The distribution of anthocyanin content per plant in F2 segregated population, 44 plants had lower anthocyanin content than WTC leaves. (PNG 272 KB)
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Supplementary file2 Supplementary Fig. S2 GO and KEGG enrichment analysis of up-regulated differentially expressed genes in ZBC. a GO enrichment analysis of up-regulated differentially expressed genes in ZBC. b KEGG enrichment analysis of up-regulated differentially expressed genes in ZBC. (PNG 582 KB)
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Supplementary file3 Supplementary Fig. S3 Evolution, gene structure and conserved domain analysis of ANS homologs in Arabidopsis and Brassica. (JPG 2599 KB)
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Supplementary file4 Supplementary Fig. S4 The protein-protein interaction network of differentially expressed genes related to anthocyanins in young leaves of ZBC and WTC. Seventeen anthocyanin-related genes were involved in the construction of the protein-protein interaction network, and the colorful balls represent different proteins and the line between proteins indicates the interaction between the two proteins. (JPG 3425 KB)
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Supplementary file5 Supplementary Fig. S5 PCR amplification of LBD39 gene agarose gel electrophoresis. a Genomic DNA amplified the LBD39 gene. b LBD39 gene was amplified by reverse transcription cDNA. (PNG 260 KB)
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Supplementary file6 Supplementary Fig. S6 Analysis of LBD39 gene expression. a Expression of LBD39 in WTC and ZBC leaf transcriptome. b The expression level of LBD39 in WTC and ZBC leaves was verified by qRT-PCR. Error bars indicate SE from three replicates (**P<0.01). (PNG 8 KB)
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Supplementary file7 Supplementary Table S1 Annotated genes have been obtained for the A3 Chromosome target segment. (XLSX 69 KB)
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Supplementary file9 Supplementary Table S3 GO enrichment analysis of differentially expressed genes in young leaves of ZBC and WTC. (XLSX 31 KB)
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Supplementary file10 Supplementary Table S4 KEGG enrichment analysis of differentially expressed genes in young leaves of ZBC and WTC. (XLSX 13 KB)
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Supplementary file11 Supplementary Table S5 Differentially expressed genes in the target region of chromosome A03 of B.rapa (XLSX 17 KB)
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Supplementary file12 Supplementary Table S6 Transcriptome sequencing and expression levels of four Chinese cabbage subspecies (XLSX 21 KB)
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Tan, C., Chen, H., Dai, G. et al. Identification and characterization of the gene BraANS.A03 associated with purple leaf color in pak choi (Brassica rapa L. ssp. chinensis). Planta 258, 19 (2023). https://doi.org/10.1007/s00425-023-04171-7
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DOI: https://doi.org/10.1007/s00425-023-04171-7