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The introgression of BjMYB113 from Brassica juncea leads to purple leaf trait in Brassica napus

BMC Plant Biology volume 24, Article number: 735 (2024) Cite this article

Abstract

The purple leaves of Brassica napus are abundant in anthocyanins, which are renowned for their role in conferring distinct colors, stress tolerance, and health benefits, however the genetic basis of this trait in B. napus remains largely unelucidated. Herein, the purple leaf B. napus (PL) exhibited purple pigments in the upper epidermis and a substantial increase in anthocyanin accumulation, particularly of cyanidin, compared to green leaf B. napus (GL). The genetic control of the purple leaf trait was attributed to a semi-dominant gene, pl, which was mapped to the end of chromosome A03. However, sequencing of the fragments amplified by the markers linked to pl indicated that they were all mapped to chromosome B05 from B. juncea. Within this B05 chromosomal segment, the BjMYB113 gene-specific marker showed perfect co-segregation with the purple leaf trait in the F2 population, suggesting that the BjMYB113 introgression from B. juncea was the candidate gene for the purple leaf trait in B. napus. To further verify the function of candidate gene, CRISPR/Cas9 was performed to knock out the BjMYB113 gene in PL. The three myb113 mutants exhibited evident green leaf phenotype, absence of purple pigments in the adaxial epidermis, and a significantly reduced accumulation of anthocyanin compared to PL. Additionally, the genes involved in positive regulatory (TT8), late anthocyanin biosynthesis (DFR, ANS, UFGT), as well as transport genes (TT19) were significantly suppressed in the myb113 mutants, further confirming that BjMYB113 was response for the anthocyanin accumulation in purple leaf B. napus. This study contributes to an advanced understanding of the regulation mechanism of anthocyanin accumulation in B. napus.

Key message

Map-based cloning and CRISPR/Cas9-based editing verified that BjMYB113 from B. juncea conferred the anthocyanin accumulation in purple leaf B. napus.

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Introduction

Anthocyanins are a class of water-soluble pigments present in the vacuolar of almost all flowering plants. Anthocyanins can be found in different plant tissues and confer shades of red, purple, and blue, depending on their molecular structure, intermolecular co-pigmentation, pH in vacuolar, and so on [1, 2]. There is growing evidence that anthocyanins not only give plant tissues distinct colors [2] but also endow plants with important physiological functions, such as photoprotection [3], antioxidant [4], salt and drought tolerance [5,6,7].

The anthocyanin biosynthesis is derived from branches of the flavonoid pathway and catalyzed by a series of enzymes, such as dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS/LDOX), anthocyanidin 3-O-glucosyltransferase (UFGT), and so on [1, 8,9,10]. These structural genes are mainly regulated by the MBW ternary complex, including R2R3-MYB transcription factors (PAP2, MYB113, and MYB114), basic helix–loop–helix transcription factors (TT8), WD40, and other transcription factors [11,12,13,14]. In general, the upregulation of these components of the MBW complex, except for MYBL2, could activate the transcription in the late anthocyanin biosynthesis pathway [15, 16]. In Arabidopsis, TT8, TTG1, and PAP2, together with other MYB transcription factors formed the MBW ternary complexes in a tissue-specific manner. The complexes could directly target and active the expression of late biosynthetic genes, such as DFR, TT19, ANS, and UFGT, thereby leading to a higher accumulation of anthocyanin levels in the vegetative tissues [8, 13].

The genus Brassica contains three allotetraploids and relevant diploid progenitors [17,18,19,20]. Although many Brassica crops exhibit color variations in different tissues, the mechanisms underlying the anthocyanin accumulation are various across B. rapa, B. oleracea, and B. juncea [21]. In B. rapa, the upregulation of BrMYB2 and BrbHLH49 in chromosome A07 could promote anthocyanin biosynthesis, resulting in the dominant purple head and stalks trait, respectively [22, 23]. The other gene in A07, BrMYBL2, was identified as a negative regulator in anthocyanin biosynthesis by repressing MBW complex activity [24, 25]. In B. oleracea, map-based cloning, overexpression, virus-induced gene silencing, as well as CRISPR/Cas9-based knockout assay confirmed that BoDFR1 accounted for the anthocyanin accumulation in pink-leaved ornamental kale and no-anthocyanin-accumulation phenotype in curly kale [26,27,28]. Consistent with the findings in B. rapa, the absence of BoMYBL2-1 expression was also responsible for purple coloration in cabbages [15]. Recent studies in B. juncea suggested that BjMYB113 was mapped to chromosome B05, and frequent gain- and loss-of-function mutations of this gene were response for leaf color variation [29, 30]. Transient expression of BjMYB113 from the purple B. juncea cultivar in Nicotiana tabacum leaves could increase anthocyanin biosynthesis [29]. Meanwhile, overexpressing BjMYB113 alleles in Arabidopsis lines also showed purple leaves and stems in comparison with green leaves and stems in wild type [30]. As far as we know, there is no direct knockout of this gene has been reported yet either in B. juncea or in B. napus.

B. napus (AACC, 2n = 38) is derived from interspecific hybridization between the diploid progenitors B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18), and introduced abundant subgenomic variation via intraspecific, interspecific and intergeneric crosses [19, 31, 32]. Overexpression of BnPAP2 from chromosome A07 or OvPAP2 from Orychophragmus violaceus could lead to the activation of anthocyanin biosynthesis genes, thereby promoting the accumulation of anthocyanins in leaves or flowers [33,34,35]. While the BnbHLH92a negatively regulates anthocyanin biosynthesis in seeds [14]. Another study in purple leaf B. napus suggested that BnAPR2 at the ends of chromosome A03, which encodes adenosine 5′-phosphosulfate reductase, is likely to be a valuable candidate gene [36]. In this research, we characterized a critical gene, BjMYB113 from B. juncea, which was connected with controlling purple leaf trait in B. napus. Moreover, knockout of this gene using CRISPR/Cas9 significantly reduced the anthocyanin biosynthesis, confirming the function of BjMYB113.

Materials and methods

Plant material and genetic analysis

The two B. napus inbred lines exhibiting purple leaves (abbreviated as PL) and green leaves (abbreviated as GL) at the seedling stage were cultivated in accordance with the methods outlined in our previous study [37]. One of the B. napus open-pollinated progeny that exhibited the purple leaf trait was derived from the interspecific hybridization between green leaf B. napus and purple leaf B. juncea. The individual plants with purple leaves were selected and backcrossed with B. napus six times. Subsequently, the lines with purple leaves were self-pollinated for six generations to generate the PL inbred lines. The inbred line GL was used as a female parent to cross with PL in order to produce F1 plants. After that, F2 populations obtained from the self-pollination of F1 were utilized for map-based cloning. The plants were grown under natural lighting at the Hunan University of Science and Technology, China.

Anthocyanin extractions and measurements

The total anthocyanin content of GL, PL, and transgenic lines was measured using the method as we described previously [10]. Firstly, the young leaves (0.1 g) were ground into powder in liquid nitrogen and then mixed with 1 mL of 95% ethanol:1.5 mol L–1 HCl (85:15, v/v) for 24 h at 4 °C in the dark with moderate shaking. Subsequently, the absorption of the aqueous phase was measured at 530 nm using Cary 60 UV–Vis (Agilent Technologies, Palo Alto, USA).

For metabolite profiling analysis, approximately 5 g of leaf tissue per line was collected, and six biological replicates of each sample were analyzed. Subsequent metabolite extraction, identification, and quantification of different types of anthocyanins were performed using a UPLC-MS/MS system (UPLC, SHIMADZU Nexera X2) at Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China). Metabolites meeting the criteria of P < 0.05 and an absolute log2FC (fold change) ≥ 1 were identified as differentially accumulated between groups.

Bulked segregant analysis

Bulked Segregant Analysis (BSA) was performed to map gene or genes responsible for the purple leaf trait. A total of 30 purple and 30 green leaf individuals from an F2 segregating population were mixed as the purple pool (F2P) and the green pool (F2G), respectively. These pools, along with the parental lines (PL and GL), were sequenced on the Illumina Novaseq 6000 platform by Shanghai Majorbio Bio-pharm Technology Co., Ltd., China. Approximately 87.8 G clean reads were obtained, and the clean reads were mapped to the Brassica napus reference genome (http://cbi.hzau.edu.cn/bnapus/) using the BWA software. SNPs between the F2P and F2G were identified by using GATK software. The ΔSNP-index was used to identify the region linked to the target gene. The raw reads were available at https://ngdc.cncb.ac.cn/gsa/, CRA013765.

Construction of genetic linkage maps

Polymorphic markers that were detected in F2P, but not in F2G were used to screen the F2 population and fine map the gene. The genetic distance in centimorgans (cM) was calculated using the Joinmap4.0 software, and a genetic linkage map was drawn with the polymorphic markers using an Excel macro, MapDraw. All the primers used in our study are detailed in Table S1.

Sequence analysis of BjMYB113 and fragments amplified by the markers

The 2 × SanTaq PCR Master Mix (Sangon Biotech, China) was used to amplify the BjMYB113 gene and flanking markers. The PCR products were recycled by SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, China), and then cloned into a pMD 18-T Vector (TaKaRa, China). After transformation into E. coli DH5α Competent Cells, AMP resistance screening was performed and 15 positive colonies were selected for LB liquid culture and sent to the Sangon Biotech company for sequencing.

The sequences of BjMYB113 and flanking markers were performed by BLAST searches of the B. juncea and B. napus genome in BRAD [38]. Phylogenetic trees of MYB113, PAP1, and PAP2 proteins were constructed using MEGA7.0 software with the Neighbor-Joining and 1000 replicated bootstrap.

Construction of the CRISPR/Cas9 vector and plant transformation

Two sgRNA sequences were designed to target the BjMYB113 gene using CRISPR-P 2.0 (http://cbi.hzau.edu.cn/CRISPR2/). The expression cassettes with target sequence were integrated into the pKSE401 CRISPR/Cas9 vector and subsequently transformed into PL via the Agrobacterium-mediated hypocotyl method. The plasmid-specific primers were designed to screen the transgenic plants. The screened T0 plants were selected and self-pollination to generate the T1 lines. The PCR products of BjMYB113 in the transgenic lines were transformed into E. coli for further Sanger sequencing. Thress independent transgenic lines, including two heterozygous lines (myb113-1, myb113-2) and one homozygous line (myb113-3) were used in subsequently study.

RNA extraction and gene expression analysis

For gene expression analysis, RNA was extracted from the fresh leaves of PL, GL, and transgenic lines using the RNA Easy Fast Plant Tissue Kit (TIANGEN, Beijing, China) following a standard protocol. A cDNA synthesis kit of Tsingke Goldenstar RT6cDNA Synthesis Kit ver.2 (Tsingke, Beijing, China) in conjunction with 1 μg of total RNA was used for reverse transcription. The internal reference gene was the β-actin (GenBank accession No. AF111812), and SYBR Premix Ex TaqII with a Bio-RadCFX96 Real-Time Detection System.

The RNA-Seq data of PL and GL at 41, 91, and 101 days after sowing which we have used in our previous study [37] were also re-analyzed using the lasted version of the B. napus reference genome [39].

Results

Phenotypic and metabolomic analysis of the purple leaf trait in B. napus

During the seedling stage, the PL displayed purple pigments in the upper epidermis and thin vein of leaves, while no obvious purple pigments were observed in the leaves of GL (Fig. 1a). The total anthocyanin content was varied at different developmental stages in the leaves of PL, and could be visually distinguished before the bolting stage [37]. Approximately 1.08 ± 0.047 mg/g anthocyanins were found in the leaves of PL at 6 weeks after sowing, which was significantly higher (P < 0.01) than that in GL (0.32 ± 0.047 mg/g, fresh weight, Fig. 1b).

Fig. 1
figure 1

Phenotypes of B. napus with purple and green leaves. a Phenotype and lower epidermis of B. napus with purple (PL), green leaves (GL), and F1 progeny. b Content of total anthocyanin accumulation in PL and GL. The error bars represent the standard error of six biological replicates. c Accumulation of different types of cyanidin, flavonoid, and petunidin in the six biological replicates of PL and GL. Asterisks indicate significant differences in a t-test (**, P < 0.01). d Differentially accumulated metabolites in the leaves between PL and GL. The purple points indicate significantly higher accumulation of metabolites and the green points indicate significantly lower accumulation of metabolites in the PL as compared with GL

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We further used the UPLC-MS/MS system to identify the types of anthocyanins and quantify their concentrations in the PL and GL. A total of 50 anthocyanin-related metabolites were identified, and 16 of them exhibited significantly higher accumulation in PL than GL, 13 anthocyanins had significantly higher concentration in the GL (Fig. 1c and 1d). Notably, significantly more cyanidins and flavonoids, including Cyanidin-3,5,3'-O-triglucoside, Cyanidin-3-O-sophoroside, Cyanidin-3-O-(6-O-p-coumaroyl)-glucoside, Cyanidin-3-O-5-O-(6-O-coumaroyl)-diglucoside were accumulated in the leaves of PL as compared with GL. While more petunidin, such as Petunidin-3-O-galactoside, Petunidin-3-O-glucoside, and Petunidin-3-O-sophoroside were found in the leaves of GL.

BjMYB113 is a strong candidate gene controlling the purple leaf trait

The PL was crossed with GL to generate F1 and F2 progeny (Table 1). The F1 displayed mauve leaves, and the F2 segregating population showed the expected Mendelian segregation ratio of 1(purple):2(mauve):1(green) (χ2 < 5.99, P > 0.05). Moreover, in the BC1 population, the expected ratio of 1:1 was also observed (χ2 < 3.84, P > 0.05), suggesting a single gene controlled the purple leaf trait (pl) in B. napus, and the trait was incomplete dominance.

Table 1 Genetic analyses of the purple leaf trait in different segregating populations
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The BSA method was performed for preliminary mapping of the pl gene (Fig. 2a). Approximately 12.17G, 10.29G, 33.13G, and 32.25G reads were generated from the PL, GL, F2P, and F2G pools, respectively. More than 99% of the clean reads were successfully aligned to the B. napus reference genome. Based on the distribution of the ΔSNP-index, the pl gene was mapped to the ends of chromosome A03, which was consistent with the findings in the previous study [36]. However, when we checked the expression value of the BnAPR2 gene using our previous RNA-Seq data [37], no significant difference was observed between the PL and GL either at 41 days, 91 days, or at 101 days after sowing (Fig. S1).

Fig. 2
figure 2

Map-based cloning of the gene controls the purple leaf trait in the F2 population. a The distribution of the average value of ΔSNP-index plotted along the 19 chromosomes of B. napus. The red line indicates the threshold line. b Fine mapping of pl using 476 segregating individuals. The maker 598–1 was developed according to the BnAPR2 at the ends of chromosome A03 as described previously [36]. c Schematic diagram of predicted genes in the pl locus. The black broad arrows represent the predicted genes that present only in the PL. The white broad arrows represent the predicted genes that absent in both PL and GL by PCR method. The BjMYB113 (BjuVB05G51160) gene was in purple broad arrow, considered as the candidate gene. d Molecular markers of BjMYB113 were used to distinguish different genotypes in the parental line and the segregating population

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To further narrow the target region, we developed polymorphic markers to genotype the individuals of the F2 population. After screening a total of 476 individuals from the F2 population, the pl gene was mapped between markers 598–1 and BJU314 (Fig. 2b). Using the other four makers, we ultimately confirmed that BJU200 and BJU201 were flanking and linked to the purple leaf trait, and the genetic distances were 0.2 cM, respectively.

Interestingly, when we sequenced the fragments amplified by the markers linked to pl, they were all mapped to the chromosome B05 of B. juncea, rather than the chromosome A03 of B. napus (Table 2). Within the mapping region of chromosome B05 from B. juncea, a candidate gene, BjMYB113, has been reported to be related to the purple leaf trait in B. juncea [30]. Thus we speculated that the introgression of BjMYB113 from B. juncea might lead to the purple leaf trait in B. napus. To further investigate this, we amplified the genomic sequences of BjMYB113 (BjuVB05G51160) and its flanking genes, based on the annotations of the Braju_tum_V2.0 reference genome [38]. Our analysis revealed that BjMYB113, as well as its flank genes, were both absent in the GL. Although genes mapped on the left flank (55.31 M -55.52 M) of BjMYB113 were also lacking in PL, the BjMYB113 and those genes on its right flank (55.56 M -55.87 M) were present in the PL (Fig. 2c). These results suggested that the gene introgression rather than the addition of the whole B05 chromosome occurred during the cultivation process PL. Importantly, the BjMYB113 gene marker showed perfect co-segregation with the phenotypes of 476 F2 individuals as expected, and BjMYB113 was only present in individuals with purple leaf but absent in individuals with green leaf (Fig. 2d). This provides compelling evidence for the association of BjMYB113 with the purple leaf trait in B. napus.

Table 2 The sequence similarities of fragments amplified by the markers in the linkage map
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The phenotype of the purple leaf trait is dependent on a functional BjMYB113

The BjMYB113 amplified in the PL was highly conserved concerning the proteins to the BjMYB113a, BjMYB113b, and BjMYB113c, but not in the subgroup of PAP1 and PAP2, indicating the potential conservation of MYB113 function in B. juncea (Fig. S2). To further confirm the function of BjMYB113, we knocked out BjMYB113 in PL plants via the CRISPR/Cas9 genome-editing system. Three specific target sites were designed in the third exons of BjMYB113(Fig. 3a). We successfully generated 3 independent transgenic lines (myb113-1, myb113-2, myb113-3) that harbored mutations in the targeted region (Fig. 3b). Specifically, the mutation in myb113-1 resulted in a 3 bases substitution, causing a single amino acid variation (from Ala to Ser) in the protein, whereas the single base deletion (-1 bp) either in myb113-2, myb113-3 were predicted to cause frameshifts and premature termination of protein translation (Fig. S3 and S4).

Fig. 3
figure 3

Creation and phenotype of BjMYB113 knockout lines. a Three target sites of exon 3 of the BjMYB113 and vector structure of CRISPR/Cas9 hosting sgRNA expression cassettes. The vertical arrow in the gene model indicates the target site. b Sequencing at the mutation sites of BjMYB113 in the T1 generation. Nucleotide indels or substitutions in three mutations are marked in purple, with details labeled at right. c Phenotype (scale bar = 2 cm) and transverse section of leaf from GL, PL, and related BjMYB113 knockout lines. d Content of total anthocyanin accumulation in PL, GL, and BjMYB113 knockout lines. The error bars represent the standard error of three biological replicates. Data were analyzed by one-way ANOVA, followed by comparisons of means using Tukey’s multiple comparisons test. Different letters indicate significant differences at p < 0.05

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The three myb113 mutants exhibited green leaf color as compared with PL, which had obvious purple leaves at the seeding stage (Fig. 3c). Transverse sections revealed purple pigments were predominantly accumulated in the adaxial epidermis and subepidermal cells of PL leaves, whereas no significant accumulation of purple pigments was observed in mutants. Given that low temperatures could promote the accumulation of anthocyanins, the total anthocyanins in the leaves of plants under 4 ℃ treatment were then extracted and measured. The average anthocyanin contents in PL were markedly higher than those in both GL and myb113 mutants (Fig. 3d). No significant difference in the content of anthocyanins was observed between the myb113-3 and GL, however, myb113-1 and myb113-2 exhibited higher content of anthocyanins than those in GL. These findings collectively underscore the essential role of BjMYB113 in anthocyanin biosynthesis in PL, with the loss function of this gene resulting in the green leaf trait.

The BjMYB113 activates the expression of anthocyanin biosynthetic genes

The MBW protein complex influences the expression of structural genes in the anthocyanin biosynthetic pathway. In our study, the BjMYB113 was highly expressed in the leaves of PL (Fig. 4a). Additionally, the RNA-Seq data showed that the other transcription factor gene TT8, which formed the MBW ternary complex together with MYB113, also showed higher expression levels in the PL as compared with GL (Fig. 4b). Moreover, the majority of MBW complex targeted genes, such as DFR, ANS, UFGT, and TT19 were significantly upregulated in the leaves of PL at 41 and 91 days after sowing. However, when the BjMYB113 was knocked out, the expression of those genes was significantly suppressed. These results collectively confirmed that that the upregulation of BjMYB113, along with its target genes in the late anthocyanin biosynthesis pathway, contributed to the promotion the anthocyanin accumulation in the leaves of PL (Fig. 4c).

Fig. 4
figure 4

The expression of BjMYB113 and anthocyanin biosynthetic genes in the parental and knockout lines. a The expression of BjMYB113 in the leaves of PL and GL. Asterisks indicate significant differences in a t-test (**, P < 0.01). b Expression of anthocyanin biosynthetic genes in the leaves of PL, GL, and knockout lines. The color scale at the left indicates Log2 transformed average FPKM values from three replicates after re-analyzing our previous RNA-Seq data. The color scale at the right indicates the relative expression of genes from three replicates using qRT-PCR methods. c The regulatory mechanism of BjMYB113 on the purple leaf trait in B. napus

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Discussion

Anthocyanins were widely known for conferring colors to plant tissues, and their contribution to plant stress tolerance and health benefits [13]. Although B. oleracea and B. juncea vegetables had varying degrees of anthocyanin accumulation and exhibited color variations in leaves [28, 40], the purple leaf B. napus and the mechanisms underlying the purple leaf trait have been rarely reported [36]. Herein, our results found that the total anthocyanins content in the PL was significantly higher than that in GL, and cyanidins were the major contributor to the purple leaf trait (Fig. 1). Although the anthocyanins profile in Brassica differed among species, the higher accumulation of cyanidins were generally observed in different types of B. oleracea and B. juncea with purple or red tissues [41]. A recent study suggested that cyanidins, especially the cyanidin 3-diglucoside-5-glucoside derivatives, were the main component of purple leaves and petunidin predominated in green leaves in B. napus [42], which was also in accord with our findings of more petunidin in GL.

In previous research, extensive genetic regulation studies of anthocyanins have been conducted in the model plant A. thaliana [12]. Notably, through map-based cloning, several genes, specifically BrMYB2 from B. rapa [23], BoDFR1, BoMYB2, and BoMYBL2-1 from B. oleracea [15, 26,27,28, 43], BjMYB113 from B. juncea [29, 44], and BnPAP2 and BnbHLH92a from B. napus [14, 34, 35] have been well characterized as significant contributors to anthocyanin accumulation. In our study, the gene controlling the purple leaf trait was mapped to the ends of chromosome A03 (Fig. 2), which was consistent with the chromosome location in the previous study, but contradictory to the identification of the BnAPR2 gene as the candidate gene [36]. Our subsequent analysis revealed several key points. Firstly, the expression level of the BnAPR2 gene showed no significant difference between the PL and GL either at 41 days, 91 days, or 101 days after sowing (Fig. S1). Secondly, the marker developed based on the BnAPR2 gene was found to be 22.9 cM away from the pl locus and did not exhibit perfect co-segregation with the observed phenotypes (Fig. 2b). Lastly, the fragments amplified by the markers linked to pl were all mapped to the B05 chromosome of B. juncea, rather than A03 chromosome of B. napus (Fig. 2c and Table 2). These results indicated that a gene from B. juncea chromosome B05, rather than BnAPR2, should be regarded as the candidate gene for purple leaf trait in our study.

Recent studies in B. juncea suggested that BjMYB113 was mapped to the chromosome B05, and ectopic expression of BjMYB113 in N. tabacum and A. thaliana both resulted in purple leaves [29, 30]. Therefore, we hypothesized that the introgression of BjMYB113 through interspecific hybridization might be responsible for the purple leaf trait in B. napus. Screening of the BjMYB113 gene marker in the 476 F2 individuals showed perfect co-segregation with the phenotypes (Fig. 2d). Moreover, knocked out BjMYB113 in PL plants via the CRISPR/Cas9 genome-editing system (Fig. 3b) could result in the obvious green leaf phenotype, absent of purple pigments (Fig. 3c), as well as the significantly less accumulation of anthocyanin (Fig. 3d) in the myb113 mutants as compared with PL (Fig. 3). These results suggested a critical role for BjMYB113 in the control of the accumulation of anthocyanin in the leave of PL. MYB transcription factors, as one of the complexes that formed the MBW ternary, played critical roles in the activation of structural genes in the anthocyanin biosynthetic pathway [11, 13]. The activation of the BoMYB2 gene could lead to anthocyanin accumulation in three purple B. oleracea cultivars [43]. Overexpression of BnPAP2 or OvPAP2 could activate the expression of anthocyanin biosynthesis-related genes, thereby promoting the accumulation of anthocyanins in both leaves or flowers [33,34,35]. In our study, we observed that BjMYB113, together with MBW complex target genes in the late anthocyanin biosynthesis pathway such as TT8, DFR, ANS, UFGT, and TT19 were significantly upregulated in the leaves of PL. Notably, the overexpression of these genes involved in positive regulatory, late anthocyanin biosynthesis as well as transport, was commonly associated with increased levels of anthocyanin accumulation in Brassica [44,45,46]. Meanwhile, the knockout of BjMYB113 leads to a significant suppression of the expression of these genes, further confirming that BjMYB113 participated in the transcriptional regulation in the anthocyanin biosynthetic pathway by positive regulatory feedback (Fig. 4). As BjMYB113 originated from interspecific hybridization between B. juncea and B. napus, further research into the formation, direct targets, and spatio-temporal activity of MBW complexes is necessary to gain a comprehensive understanding of the transcriptional mechanisms that regulate anthocyanin biosynthesis.

Availability of data and materials

The raw reads of BSA-Seq are available in the Genome Sequence Archive in National Genomics Data Center (https://ngdc.cncb.ac.cn/gsa/) under accession number CRA013765. The RNA-Seq raw data during the current study are available under accession number CRA004724. All data generated or analyzed during this study are included in this published article and its supplementary information files.

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Funding

This work is supported by the National Natural Science Foundation of China (U19A2029), the Science and Technology Innovation Program of Hunan Province (2023JJ40279,2023RC1077), the Scientific Research Fund of Hunan Provincial Education Department (21B0490), and the Foundation of State Key Laboratory of Utilization of Woody Oil Resource (GZKF202204).

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Authors and Affiliations

  1. Hunan Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, School of Life and Health Science, Hunan University of Science and Technology, Xiangtan, 411201, China

    Dawei Zhang, Hongfeng Zhou, Dinggang Zhou, Jinfeng Wu & Lili Liu

  2. Yuelushan Laboratory, Changsha, 410128, China

    Dawei Zhang, Jinfeng Wu, Lili Liu, Yiming Guo, Tonghua Wang & Mingli Yan

  3. Hunan Research Center of Heterosis Utilization in Rapeseed, Crop Research Institute, Hunan Academy of Agricultural Sciences, Changsha, 410125, China

    Dawei Zhang, Yiming Guo, Tonghua Wang & Mingli Yan

  4. College of Life Sciences, Ganzhou Key Laboratory of Greenhouse Vegetable, Gannan Normal University, Ganzhou, 341000, China

    Chen Tan & Daozong Chen

  5. College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Xianhong Ge

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  1. Dawei Zhang
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Contributions

ZDW, YML conceived and designed all experiments. ZHF, ZDG, WJF, LLL, TC and CDZ performed the experiments. GYM and WTH cultivated the plant material. ZDW analyzed the data and wrote the manuscript. GXH revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Mingli Yan.

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Zhang, D., Zhou, H., Zhou, D. et al. The introgression of BjMYB113 from Brassica juncea leads to purple leaf trait in Brassica napus. BMC Plant Biol 24, 735 (2024). https://doi.org/10.1186/s12870-024-05418-5

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BMC Plant Biology

ISSN: 1471-2229

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