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Article

Expression Pattern Analyzation and Regulation Genes Identification on the Purple Phenotype in the Different Tissues of the Purple Pak-choi and Zicaitai

by
Yating Zhu
†,
Xia Wang
†,
Xiuping Tu
,
Shujiang Zhang
,
Shifan Zhang
,
Hui Zhang
,
Rifei Sun
,
Xiuxiu Xu
,
Xinyu Gao
,
Guoliang Li
* and
Fei Li
*
State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(2), 109; https://doi.org/10.3390/horticulturae11020109
Submission received: 25 November 2024 / Revised: 2 January 2025 / Accepted: 17 January 2025 / Published: 21 January 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Browse Figures
Versions Notes

Abstract

:
Among the Brassica species in China, including Chinese cabbage, pak-choi, caixin, zicaitai, and wucai, Brassica rapa plays an important role in vegetable production. Purple resources from the species itself are scarce. It is worth noting that the tissue positions expressing a purple phenotype vary greatly between purple pak-choi and zicaitai. In this study, the genetic patterns of the purple phenotype were analyzed in purple pak-choi and zicaitai, and the F1 showed purple leaves and green stems, which indicated that purple traits in the leaves and stem were inherited independently. In conjunction with field identification, RNA-seq was used to sequence the transcriptomes of the purple expression sites of purple pak-choi, zicaitai, and their F1. The high expression of the regulatory genes Dark_pur and BrTT8 affected the purple color of pak-choi and caused the high expression of structural genes of the anthocyanin metabolism pathway and the accumulation of anthocyanins in leaves. The regulatory genes BrPAP2 and BrTT8 affected the purple color of zicaitai stems, were significantly upregulated, and caused high expression of related structural genes, leading to the accumulation of anthocyanins in the stem epidermis. This suggested that BrPAP2 and Dark_pur were both R2R3-MYB transcription factors, which were tissue-specific for the regulation of purple color traits in B. rapa. They also had a gene epistatic effect, which influenced the expression of purple traits in the F1. The gene MYBL2 was highly expressed in all purple tissue sites. The present study on the regulatory genes of the purple phenotype of zicaitai and purple pak-choi provides a theoretical basis for revealing the influence of purple traits on B. rapa leaves and stems, and it may lay the foundation for the selection and breeding of purple vegetables of B. rapa.

1. Introduction

Anthocyanin is an important secondary metabolite in plants, and anthocyanin-rich vegetables and fruits are increasingly favored by consumers [1,2,3]. The molecular mechanisms of anthocyanin biosynthesis and accumulation have been extensively studied in many plants. It was believed that anthocyanin synthesis is influenced by structural genes, which, in dicotyledonous plants, include early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs) [4,5,6,7,8]. The anthocyanin synthesis pathway is divided into three stages, and synthesis mainly occurs through the regulation of genes encoding enzymes in the phenylpropane metabolic pathway. First, the formation of 4-coumaroyl-CoA, the main precursor of anthocyanin synthesis, is catalyzed by phenylalanine through phenylalanine deaminase (PAL), cinnamic acid 4-hydroxylase (C4H), and 4-coumarate:coenzyme aligase in the flavonoid metabolic pathway [9]. The second step, a key reaction in anthocyanin metabolism, generates dihydroflavonol from 4-coumaroyl-CoA and malonyl-CoA. This step is regulated by chalcone synthase (CHS) and flavanone-3-hydroxylase (F3H) [10,11]. The third step, the conversion of colorless dihydroflavonol into colorless anthocyanins and, thus, the synthesis of unmodified anthocyanins, is regulated by two enzymatic activities: dihydroflavonol reductase (DFR) and anthocyanin synthase (ANS/LDOX) [12,13,14]. Among the structural genes associated with the anthocyanin synthesis pathway, CHS is the key enzyme that initiates flavonoid biosynthesis, and F3’H and F3’5’H, as the major enzymes in the anthocyanin synthesis pathway, collectively contribute to anthocyanin diversification by regulating the hydroxylation pattern of flavonols, synergizing with other enzymes, and responding to changes in environmental factors [15,16]. In the last few decades, the regulation of anthocyanin biosynthesis with structural genes has been relatively clear; current studies have shown that the biosynthetic pathway is relatively conserved among different species, while the regulatory genes of anthocyanin biosynthesis in different organs and tissues vary. MYB transcription factors (MYB TFs), together with basic helix–loop–helix (bHLH) and WD40/WDR regulators, usually form MBW complexes and affect the anthocyanin levels by regulating the expression of the corresponding biosynthetic genes [17,18,19,20,21].
B. rapa is one of the most important genera in Cruciferae. There are few purple germplasm resources of the Brassica genus. Now, there are many common cultivars in production, such as purple cabbage, purple leaf mustard, and purple turnip (zicaitai). In recent years, breeding researchers have been increasingly concerned with the creation of purple vegetables. Now, inter-species hybridization of B. rapa is used to obtain purple resources by hybridizing green-heading Chinese cabbage with zicaitai to obtain a new purple germplasm [22], the inner leaves of which have a purple color. Using distant hybridization and an embryo rescue technique on purple-leaf B. juncea and green-heading Chinese cabbage [23,24], a new germplasm for purple-heading Chinese cabbage with leaves that were enriched in anthocyanins was obtained.
Recent studies have shown that in the bHLH family, BrTT8 was considered a candidate gene for purple Chinese cabbage [25]. The deletion/insertion of the bHLH 49 promoter region was significantly associated with the color traits of zicaitai stems [22,26], and BrEGL3.1 and BrEGL3.2 are homologs of Arabidopsis thaliana gene AtEGL3, which is a key gene controlling the accumulation of anthocyanin in the stems of zicaitai [27]. Moreover, in the genes of the MYB family, R2R3-MYB transcription factors appear to be important in the regulation of anthocyanin biosynthesis. Overexpression of the R2R3-MYBs of AtPAP1, AtPAP2, AtMYB113, and AtMYB114 led to a large accumulation of anthocyanins in Arabidopsis seed [17].
A transcript numbered c3563g1i2 from B. juncea was found to be highly expressed in the new purple germplasm of Chinese cabbage; it encoded the R2R3-MYB transcription factor, which is a key gene regulating the change in leaf color from green to purple [28]. This regulatory gene comes from B. juncea chromosome B05 [29]; its gene function was verified through translation into Chinese cabbage in our previous study, and this gene was named Dark_pur [30]. A study on the BrMYB2 gene showed that structural variations in the BrMYB2 gene led to stems of zicaitai with a purple phenotype [31]. Some researchers have suggested that BrMYB2 is a key gene controlling purple-heading Chinese cabbage [32,33,34,35]. In addition, the R3-MYB transcription factor of the BrMYBL2.1 gene is a negative regulator of anthocyanin biosynthesis in zicaitai shoots [36]. These studies showed that the MYB and bHLH transcription factors are closely related to anthocyanin synthesis in Brassica crops and are relatively complex.
In the early stage of this project, a series of new purple germplasms, including that of purple pak-choi, were obtained through a distant cross between B. rapa and purple B. juncea, in which only the leaves were enriched in anthocyanins and showed a purple color [37]. The key genes regulating the purple leaf trait were derived from the B genome of B. juncea. This was in contrast to the traditional zicaitai cultivar, which showed the accumulation of anthocyanins in the stalk epidermis. It is speculated that anthocyanin accumulation with different tissue specificity can be achieved by using different regulatory genes.
In order to further analyze the influence of key regulatory genes in the anthocyanin metabolic pathway on the anthocyanin biosynthesis of B. rapa, we conducted a phenotype survey and genetic analysis of purple traits in purple materials with different regulatory backgrounds, using zicaitai and purple pak-choi as parents and their F1 to explore the effects of important regulatory genes of purple traits from different sources on the metabolic regulation of anthocyanin biosynthesis in B. rapa.

2. Materials and Methods

2.1. Plant Materials

The experimental materials used were all provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Zicaitai ‘1799’, caixin ‘1608’, and green pak-choi ‘0913’ were all high-inbreeding lines, and purple pak-choi ‘1395’ was a DH line. Three populations were obtained. First, F1-1 was obtained from ‘1395’ × ‘0913’, F2-1 was obtained from the self-pollination of F1-1, and BC1 was obtained from F1-1 × ‘0913’. Second, F1-2 was obtained from ‘1799’ × ‘1608’. Third, F1-3 was obtained from ‘1395’ × ‘1799’, and F2-3 was obtained from the self-pollination of F1-3 (F1-3 was named ‘1396’).

2.2. Investigation and Analysis of the Purple Phenotype in the Above Three Populations

In the cotyledon stage, true leaf stage, and large leaf stage, the purple phenotype was investigated in the three populations described above; the investigation included the front side of the leaves, the back side of the leaves, the color of the leaf veins, the color of the leaf petioles, and the main stem of the plants. In addition, the depth of purple and the degrees of light and dark were investigated and analyzed.

2.3. RNA Extraction and Illumina Sequencing

The third population was selected to analyze the expression pattern of the purple phenotype, and purple pak-choi ‘1395’, zicaitai ‘1799’, and F1-3 (‘1396’) were selected to extract RNA from the leaves and stems at the adult stage. Leaves were selected from young and distinctly colored tissues; stems were taken only from the young epidermis that showed color, and the middle layer and vascular column were partially removed. There were three biological replicates (1395-Y1 to -Y3 standing for the purple leaf of pak-choi, 1395-G1 to -G3 standing for the green stem of pak-choi, 1799-Y1 to -Y3 standing for the green leaf of zicaitai, 1799-G1 to -G3 standing for the purple stem of zicaitai, 1396-Y1 to -Y3 standing for the purple leaf of F1-3, and 1396-G1 to -G3 standing for the green stem of F1-3). RNAs were extracted from the leaf samples using the TransGen Biotech RNA kit, and the total amount of RNA in a single sample was 1 ng. Eighteen sequencing libraries were constructed according to the sequencing process of the Illumina platform and entrusted to Beijing Biomarker Technologies to carry out second-generation Illumina Hiseq 2500 high-throughput transcriptome sequencing. After the samples passed the quality test, cDNA libraries were constructed. Clean data were obtained by filtering the raw data and compared with the B. rapa reference genome (Brara Chiifu v3.0 version, http://Brassicadb.cn, accessed on 17 February 2024).

2.4. RNA-Seq Analysis

The raw data from transcriptome sequencing were compared with the Brara Chiifu v3.0 reference genome, and the gene expression levels were normalized using the fragments per kilobase of transcript/million mapped reads (FPKM) method.
Experimental and control DEGs were analyzed using DESeq2 with screening criteria of FDR < 0.05 and FC ≥ 2. DEGs annotated according to the GO database were enriched and analyzed using TopGO with FDR < 0.05. KEGG enrichment analysis was performed according to KOBAS (2.0) with KS < 0.05 for p-value correction.

2.5. RT-PCR Analysis and Statistical Analysis

The differentially expressed genes were screened from the transcriptome analysis, and the RNA samples were reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China); the Actin gene of B. rapa was used as an internal reference for qRT-PCR using the SYBR Green Pro Taq, and the relative expression levels were calculated using the 2−ΔΔCT method with qRT-PCR using the SYBR Green Pro Taq HS Premix qPCR Kit (Accurate Biology, Changsha, China) and the CFX-96 System (BIORAD, Hercules, CA, USA).

3. Results

3.1. Investigation and Analysis of the Purple Phenotype in Various Genetic Populations

In the first population, F1-1 was obtained from purple pak-choi ‘1395’ × green pak-choi ‘0913’, F2-1 was obtained from the self-pollination of F1-1, and BC1 was obtained from F1-1 × ‘0913’ (Figure 1A). As we reported previously [38], the segregation ratio of purple leaves to green leaves in F2-1 was 3:1, and the segregation ratio of purple leaves to green leaves in BC1 was 1:1, which indicated that the purple-leaf trait in pak-choi was regulated by a pair of dominant genes. In the second population, F1-2 was obtained from zicaitai ‘1799’ × caixin ‘1608’; the leaves of F1-2 showed a green color, and the stem was light purple (Figure 1B), which indicated that the purple-stem trait of zicaitai was incompletely dominant. In the third population, F1-3 (‘1396’) was obtained from purple pak-choi ‘1395’ × zicaitai ‘1799’, and F2-3 was obtained from the self-pollination of F1-3. It was shown that the leaves of F1-3 were purple (Figure 1C), and 208 plants of F2-3 were surveyed. The segregation ratio of purple leaves to green leaves in F2-3 was 3:1 (Table 1), which indicated that the purple-leaf trait was regulated by one pair of dominant genes, making this consistent with the results of the genetic analysis of the leaf color of pak-choi. The stem color of F1-3 was green, and the stem color of F2-3 was green, but the purple–green segregation occurred in shortened stems (purple to green = 4:9) (Table 2), which was not in accordance with the standard ratio in Mendelian inheritance law, indicating that the inheritance of stem color may involve complex mechanisms, such as polygenic inheritance, incomplete dominance, gene interactions, or environmental factors.

3.2. RNA-Seq Data Quality Control and GO Classification and Enrichment Analysis for Differential Genes

In total, 127.92 Gb of clean data were obtained, and the clean data of all samples reached 5.81 Gb; the Q30 base percentage was above 85%, and the GC content was above 46% (Table S1). All of the samples met the requirements for mutation detection and trait gene localization. Analysis of the sample clustering heat map and PCA showed that the purple phenotype expression patterns of the two parents were very different, with the F1-3 transcriptome gene expression pattern being intermediate between the two parents (Figure 2A,B). Comparative two-by-two data analysis of leaf tissues and stem parts of zicaitai ‘1395’, purple pak-choi ‘1799’, and F1-3 ‘1396’ in the same period and at the same position (Figure 2C) was used to screen a large number of differentially expressed genes that may be involved in multiple biological processes, molecular functions, and cellular components for the regulation of genes in the anthocyanin metabolic pathway.
The majority of GO classifications belonged to biological processes when analyzed by grouping parental coloring sites (1395-Y_vs._1799-Y, 1395-G_vs._1799-G) and by grouping parental material with different site comparisons (1395-Y_vs._1395-G, 1799-Y_vs._1799-G) (Figure 3). Enrichment analyses based on different comparisons clearly showed the same significant enrichments: Cellular, metabolic, and bioregulatory in the biological process classification; cellular components were significantly enriched in the cellular anatomical entity, intracellular, protein-containing complex, etc. The main significant enrichment in molecular functional classification is to binding sites, catalytic reactions, etc. Anthocyanins are synthesized in the cytoplasm and catalyzed by GST to form glutathione crosslinking complexes, which are then recognized by MRP on the vesicular membrane and transported across the membrane to the vesicles, a process that is closely related to metabolism, biological regulation, and catalytic reactions.

3.3. KEGG Classification Enrichment Analysis for Differential Genes Involved in Anthocyanin Biosynthesis

Based on the transcriptome data analysis, the zicaitai ‘1395’, purple pak-choi ‘1799’, and F1-3 ‘1396’ anthocyanin biosynthesis pathways were mapped in the leaves and stem epidermis. In leaves, the analysis showed that only Br4CL1 and Br4CL3 were highly expressed among the phenylpropane-biosynthesis-related genes in purple pak-choi, and among the BrPAL family genes, only BrPAL1.1 was highly expressed, while the CHS, CHI, F3H, F3’H, and FLS genes were highly expressed among early biosynthetic genes (EBGs). In particular, the CHI and FLS genes had the highest expression; the main role of early structural genes is to regulate the production of pro-anthocyanidins and anthocyanidins (Figure 4A, Table S2). Among the late biosynthetic genes (LBGs), DFR, ANS, and UGT were highly expressed, and these genes converted colorless anthocyanin glycosides into stable-colored anthocyanins, which were associated with leaf anthocyanin enrichment. The Br4CL1, Br4CL3, BrCHI, BrUGT78D2, BrUGT75C1, and BrANS1 genes were highly expressed in the leaf tissues of purple pak-choi. It was hypothesized that these six genes are the main structural genes involved in anthocyanin biosynthesis in purple pak-choi leaf tissues. In the leaves of zicaitai, it was shown that the BrPAL3.2 and Br4CL2.1 genes, which are related to phenylpropane biosynthesis, were expressed to a certain extent, while the other genes were not expressed or lowly expressed, so the leaf color of zicaitai was green. In the leaves of F1-3, it was shown that the genes related to phenylpropanoid biosynthesis were highly expressed, except for the Br4CL1 and Br4CL3 genes; in particular, PAL was highly expressed, and the synthesis reaction of phenylpropanoid was active, making this different from both parents. Among the EBGs, CHS and F3H were expressed to a certain extent, but the expression of the CHI and FLS genes was very low, which showed that the synthesis level of anthocyanin glycosides in F1-3 leaves was between that of the two parents. In the LBGs, except for the BrUGT78D2 gene, which had very low expression, all other genes were expressed to some extent, and although the synthesis of phenylpropanes was active in the early stage of F1-3, the final synthesis of colored-anthocyanin-related genes was lower than that in the parental purple pak-choi. The expression level of the genes related to the synthesis of colored anthocyanins was lower than that of the parental purple pak-choi, which was consistent with the fact that the purple color of its leaves was not as deep as that of the parental purple pak-choi.
Analysis of the stem tissue data showed that in zicaitai, among the genes related to phenylpropane biosynthesis, all structural genes were highly expressed, except for the BrPAL3.1 gene and BrPAL3.1 gene in the BrPAL family. The CHS, CHI, F3H, and F3’H genes, which belong to the group of EBGs, were highly expressed, with the highest expression being that of CHI, F3H, and F3’H, but there was very low expression of FLS (Figure 4B, Table S3). The FLS gene mainly induced the production of flavonols, DFR induced the synthesis of anthocyanins, and FLS and DFR were competitive. The low expression of FLS and high expression of DFR in the stem tissue also verified that FLS induced the synthesis of anthocyanins. Among LBGs, DFR, ANS, and UGT were highly expressed in zicaitai, indicating that the expression of the reaction level of the conversion of colorless anthocyanin glycosides into stable colored anthocyanins was high and that the epidermis of the stem of zicaitai was enriched with anthocyanins. The analysis showed that a total of 11 structural genes were highly expressed in the stem epidermis of zicaitai—BrPAL1.1, BrPAL2.1, Br4CL1, Br4CL2.1, BrCHS1, BrF3H1, BrDFR, BrANS1, BrUGT79B1.1, BrUGT75C1, BrUGT78D2—and they are hypothesized to be major structural genes involved in anthocyanin biosynthesis in the stem epidermis of zicaitai. In purple pak-choi, only the genes related to phenylpropane biosynthesis, BrPAL3.1 and BrPAL3.2, were highly expressed, and BrCHI was expressed to a certain extent, while the other structural genes were neither expressed nor lowly expressed, which should be the reason for the green coloration of the purple pak-choi stems. The analysis of the stem tissue data of F1-3 showed that only FLS was highly expressed among the EBGs, and there was some expression of the synthetic flavonol reaction; the expression of the rest of the structural genes was very low, which indicated that flavonols were synthesized in the stem epidermis of F1-3, but anthocyanins were not synthesized, and their expression levels were more consistent with that in the parents of purple pak-choi ‘1799’. The incomplete dominant trait of the purple stem color of zicaitai ‘1395’ did not gain expression in F1-3.
The heat map of anthocyanin structural gene expression in the purple positions of both parents was analyzed (Figure 5). The expression of phenylpropanoid-biosynthesis-related genes (PAL, C4H, 4CL) was low in both parents, and there were differences in gene expression between parents starting from the EBG CHS; the highest expression of CHI and DFR was found in zicaitai. CHI is related to the flavonoid synthesis pathway and phenylpropanoid pathway, and DFR catalyzes the dihydroflavonol production of colorless anthocyanins. DFR catalyzes the generation of colorless anthocyanins from dihydroflavonol. Compared with the two parents, the expression of genes for the epidermal structure of zicaitai was generally higher than that of purple pak-choi leaves, which was consistent with the more intense purple color of zicaitai.

3.4. Expression Analysis of Transcription Factors Related to Anthocyanin Synthesis

A predictive analysis of TF families in transcriptome data based on the plantTFDB database (http://planttfdb.gao-lab.org/, accessed on 11 May 2024) was conducted, and this was combined with the KEGG database to demonstrate that a total of 255 MYB-TFs were enriched, of which 13 MYB-TFs were associated with anthocyanin metabolism (Table 3). Plotting these 13 MYB-TFs’ expression on a heat map (Figure 6A), it was noted that three MYB transcription factors, the Drak_pur, BraA07g035710.3C, and BraA07g032100.3C genes, were highly expressed in the purple positions. The Drak_pur gene was highly expressed in purple pak-choi and F1-3 plants, and this is a key gene in the regulation of purple traits in leaves. The BraA07g032100.3C (BrPAP2) gene was highly expressed in zicaitai stems and was closely related to their purple color. This gene may be a stem-color-regulating gene in zicaitai. We noticed that the BraA07g035710.3C (BrMYBL2.1) gene showed different expression levels in purple and green tissues, and the expression in purple tissues was higher than that in green tissues.
In this study, we analyzed the expression levels of the highly expressed anthocyanin-related ‘MBW complex’ identified in the materials (Figure 6B). In the leaves of purple pak-choi ‘1799’, the expression levels of Dark_pur, BrMYBL2.1, and BrTT8 were higher. In the stem epidermal positions of zicaitai ‘1395’, there was higher expression of BrMYBL2.1, BrPAP2, and BrTT8. In F1-3 plants, the leaf color was purple, and the expression of structural and regulatory genes related to anthocyanin synthesis in the transcriptome analysis was more consistent with that in purple pak-choi ‘1799’, which may have been due to the activation of the anthocyanin metabolism pathway in the leaf area due to the high expression of Dark_pur and its binding to BrTT8. In the stem epidermis of F1-3, the expression of BrMYBL2.1, BrMYBL2.2, BrEGL3.1, and BrEGL3.1 was consistent with that of purple pak-choi ‘1799’. The expression of BrTT8 was between that in the two parents, and the expression of BrGL3 was slightly higher than that in the two parents. The expression of genes that regulate anthocyanin synthesis in the stem epidermis of F1-3 was more complex, and the overall expression was similar to that of purple pak-choi ‘1799’. The F1-3 stem positions showed a green color. Whether these two MYB TF genes competed with the binding domain of BrTT8 to affect the metabolic synthesis of anthocyanins was not clear, and further experiments were needed to prove it.

3.5. Differentially Expressed Genes Reverified with qRT-PCR

In the above expression heat map, we selected eight structural and regulatory genes related to anthocyanins that had significant expression differences between the parents; they were verified with qRT-PCR and were found to be consistent with the RNA-seq results (Figure 7, Table S4). Anthocyanin-synthesis-related structural genes and transcription factors were highly expressed in purple pak-choi. In the F1-3 individuals, the expression of these genes was overall lower than that in purple pak-choi. Analyzing the stem epidermal tissues, zicaitai stems were enriched in anthocyanins, and their structural genes and transcription factors related to anthocyanin synthesis showed high expression. The high expression of transcription factors—BrMYBL2.1, BrPAP2, and BrTT8—affected the high expression of the upstream structural genes BrCHS1 and BrCHI, and the high expression of the downstream BrDFR and BrANS1 genes resulted in anthocyanin glycosides accumulating in zicaitai stems.

3.6. qRT-PCR Analysis of High-Expression-Level Genes

BraA07g035710.3C (BrMYBL2.1) is a homolog with AtMYBL2 in Arabidopsis. This gene is R3-MYB but is positively associated with purple traits. To further validate the transcriptome results, the BrMYBL2.1 gene was cloned in purple and green material (Table S5). This gene has two bases mutated in the intron, resulting in non-synonymous mutations in the amino acid sequence. Dark_pur was identified as a purple regulatory gene in purple pak-choi in our previous study. BraA07g032100.3C (BrPAP2) was highly expressed only in purple stem epidermal tissues, and it was hypothesized that this gene was closely related to the production of anthocyanins in the stem epidermis. In order to verify this result, two additional high-generation inbred lines of zicaitai were supplemented as experimental materials for the qRT-PCR of BrPAP2 (Figure 8). This gene showed low expression in the leaves and high expression in both the stem epidermis and ovaries, which further indicated that this might be a key gene for anthocyanin synthesis in colored stems. Dark_pur and BraA07g032100.3C (BrPAP2) were both R2R3-MYBs, and they had tissue-specific expression. Dark_pur was highly expressed only in purple leaves, and BrPAP2 was highly expressed only in purple stems.

4. Discussion

This study focuses on the analysis of two unique purple B. rapa varieties, purple pak-choi and zicaitai, which are notable for the purple coloration of their leaves or stalks. Crucially, the purple regulatory genes of these two purple B. rapa varieties originated from different genetic resources. The purple-trait regulatory gene of purple pak-choi, Dark_pur, was introduced from B. juncea; its trait of purple leaf color followed a strict Mendelian pattern of inheritance and was controlled by a pair of dominant genes. In contrast, the purple regulatory gene BrPAP2 in zicaitai was derived from the A genome of the B. rapa itself, and the purple trait in the stalk area showed an incomplete dominant inheritance. The F1-3 generation again showed Mendelian inheritance regularity in the trait of purple leaf color, which was regulated by a dominant pair of genes. However, in terms of the stalk color, the F1-3 generation was uniformly green, suggesting that the BrPAP2 gene may have been modified or affected by other genetic factors in the expression of the purple stalk trait, resulting in its failure to be fully expressed in the F1 generation. To gain a deeper understanding of the mechanism of action of these purple-regulating genes and their expression in the F1 progeny of the crosses, we performed transcriptome analyses of purple pak-choi, zicaitai, and the F1 generation. Transcriptome analysis revealed that the structural genes of the anthocyanin metabolic pathway were expressed at approximately the same level, whereas there were differences in the regulatory genes.
In zicaitai, BrMYBL2.1, BrPAP2, and BrTT8 were highly expressed. In Arabidopsis, MYBL2 functions as a negative regulator of flavonoid biosynthesis, and MYBD increases anthocyanin accumulation by repressing MYBL2 expression [38,39,40,41,42]. qRT-PCR was combined with transcriptome analysis of purple- and green-leaf Chinese cabbage, showing that the expression of BrMYBL2 was significantly higher in purple leaves than in green leaves [43], and BraMYBL2 was transcribed at higher levels in purple Chinese cabbage than in green cabbage [39,44,45,46]. Previous studies on purple-stem-regulating genes also suggested that BrMYBL2.1 was a homolog of AtMYBL2. BrMYBL2.1 is a negative regulator of anthocyanin biosynthesis [22,36]. However, there was a new finding in the analysis of our experimental results: BrMYBL2.1 was R3-MYB but was positively correlated with anthocyanin content. Cloning of the BrMYBL2.1 gene in the purple/green material revealed that two bases on the third exon of the purple material were mutated, leading to changes in the amino acid sequence; ultimately, BrMYBL2.1 positively regulated anthocyanin synthesis. In addition, another study indicated that BrMYBL2 was positively correlated with anthocyanin content, which was consistent with our research results [47]. This may be unique to B.rapa. For BrPAP2, the purple stem trait of zicaitai was positively correlated with BrPAP2, as analyzed using transcriptome sequencing and qRT-PCR results. This gene is homologous to Arabidopsis AtPAP2, which encodes an MYB75 transcription factor that plays a key role in anthocyanin biosynthesis in Arabidopsis thaliana. Both knock-out mutants and knock-in mutants of AtPAP2 were defective in anthocyanin coloring in seedlings [48,49,50,51]. Based on the experimental results and reports in the literature, we concluded that the purple stem trait of zicaitai may be controlled by the BrPAP2 gene.
BrMYBL2.1, BrTT8, and Dark_pur were highly expressed in purple pak-choi. According to the results of the previous study, the purple color trait of purple pak-choi leaves is controlled by a dominant gene, Dark_pur, derived from mustard, and this encodes the R2R3-MYB transcription factor. Meanwhile, BrTT8 is highly expressed in purple leaves. MYB interacts with bHLH and WD40 to form the MBW complex, which regulates anthocyanin biosynthesis in plants. Dark_pur and BrPAP2 are R2R3-MYBs; both of them show tissue-specific expression, and they are highly expressed only in purple areas. Based on the results in Arabidopsis, it is possible that BrTT8 is positively regulated by R2R3-MYB [28,52,53,54]. The different MYB-TFs bound to BrTT8 as an important bridge in the MBW complex affect the differential expression of structural genes in the regulation of the anthocyanin metabolism pathway. Two different sources of purple parents with dominant or incomplete dominance for the purple trait showed significant differences in the expression of genes in the anthocyanin regulatory pathway, which resulted in different anthocyanin accumulation in leaves and stems. In the F1 generation, both Dark_pur and BrPAP2 regulatory genes were present, and they may compete with each other for the TT8 binding domain. This competition may result in the F1 generation showing an incompletely dominant purple trait with suppressed anthocyanin synthesis at the stem site. In the leaf site of the F1 generation, Dark_pur and BrTT8 were highly expressed, and these regulatory genes activated the anthocyanin synthesis pathway to express purple color in the leaves.
In summary, this study analyzed the differential expression levels of important anthocyanin regulatory genes and anthocyanin metabolism pathways in purple resources of B. rapa, and the purple color phenotype of B. rapa was molecularly resolved, which provides a theoretical reference for the creation of resources of distant hybrids in Brassica crops, homologous recombination of ABC genomes, and the study of molecular mechanisms for the innovation of new germplasms.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11020109/s1—Table S1: Transcriptome sequencing data quality and output statistics; Table S2: Expression of structural genes of the anthocyanin biosynthesis pathway in stems; Table S3: Expression of structural genes of the anthocyanin biosynthesis pathway in leaves; Table S4: Sequence of BrMYBL2.1 gene cloning primers; Table S5: qRT-PCR primers.

Author Contributions

Y.Z. and X.W. drafted the manuscript; Y.Z., X.W., X.T., S.Z. (Shujiang Zhang), S.Z. (Shifan Zhang), H.Z., R.S., X.X., X.G., G.L. and F.L. performed the experiments; G.L. and F.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Beijing Rural Revitalization Agricultural Science and Technology Project (NY2401140224), the National Natural Science Foundation of China (32172562), the China Agriculture Research System (CARS-23-A-14), the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS), the National Key Research and Development Program of China (No. 2022YFD1200805), and the Basic Research Funds for Central Public Welfare Research Institutes (No. IVF-BRF2024011). This work was performed at the State Key Laboratory of Vegetable Biobreeding, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China, and the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, Beijing, China.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phenotypic investigation: (A) female purple pak-choi (1395), paternal green pak-choi (0913), F1-1 (1395 × 0913)with purple leaves; (B) female zicaitai (1799), paternal caixin (1608), F1-2 with a light purple stem (1799 × 1608); (C) female purple pak-choi (1395), paternal zicaitai (1799), F1-3 (1396) with purple leaves and a green stem (1395 × 1799).
Figure 1. Phenotypic investigation: (A) female purple pak-choi (1395), paternal green pak-choi (0913), F1-1 (1395 × 0913)with purple leaves; (B) female zicaitai (1799), paternal caixin (1608), F1-2 with a light purple stem (1799 × 1608); (C) female purple pak-choi (1395), paternal zicaitai (1799), F1-3 (1396) with purple leaves and a green stem (1395 × 1799).
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Figure 2. (A) Clustering heat map of different samples for RNA-seq. The heat map shows three biological replicates per sample, with red indicating high correlation and blue indicating low correlation. (B) RNA-seq sample grouping principal component analysis (PCA) plots showing pairwise differences between samples of different colorings and sites. (C) Histograms of differentially expressed genes obtained through pairwise comparisons between the three varieties. Differential expression analysis was performed based on different materials from the same site.
Figure 2. (A) Clustering heat map of different samples for RNA-seq. The heat map shows three biological replicates per sample, with red indicating high correlation and blue indicating low correlation. (B) RNA-seq sample grouping principal component analysis (PCA) plots showing pairwise differences between samples of different colorings and sites. (C) Histograms of differentially expressed genes obtained through pairwise comparisons between the three varieties. Differential expression analysis was performed based on different materials from the same site.
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Figure 3. (A–D) Annotation of 1395-Y_vs._1799-Y, 1395-G_vs._1799-G, 1395-Y_vs._1395-G, and 1799-Y_vs._1799-G in GO enrichment analysis. The horizontal coordinates show the three major parallel classifications performed for differential genes, and the vertical coordinates on the left side indicate the number of genes. The dark fill indicates upregulation of differential genes, and the light fill indicates the downregulation of differential genes.
Figure 3. (A–D) Annotation of 1395-Y_vs._1799-Y, 1395-G_vs._1799-G, 1395-Y_vs._1395-G, and 1799-Y_vs._1799-G in GO enrichment analysis. The horizontal coordinates show the three major parallel classifications performed for differential genes, and the vertical coordinates on the left side indicate the number of genes. The dark fill indicates upregulation of differential genes, and the light fill indicates the downregulation of differential genes.
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Figure 4. (A) Expression of the genes in the anthocyanin metabolism pathway analyzed using KEGG enrichment in leaf parts. (B) KEGG enrichment analysis of the expression of genes in the anthocyanin metabolism pathway at stem sites.
Figure 4. (A) Expression of the genes in the anthocyanin metabolism pathway analyzed using KEGG enrichment in leaf parts. (B) KEGG enrichment analysis of the expression of genes in the anthocyanin metabolism pathway at stem sites.
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Figure 5. Heat map of gene expression for pak-choi leaves and the zicaitai stem structure.
Figure 5. Heat map of gene expression for pak-choi leaves and the zicaitai stem structure.
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Figure 6. (A) Heat map of the expression of 13 MYB-TFs at leaf and stem sites. (B) Important MBW analysis of anthocyanin synthesis.
Figure 6. (A) Heat map of the expression of 13 MYB-TFs at leaf and stem sites. (B) Important MBW analysis of anthocyanin synthesis.
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Figure 7. (A) qRT-PCR and RNA-seq data on gene expression patterns related to anthocyanin biosynthesis in the leaf epidermis. (B) qRT-PCR and RNA-seq data on gene expression patterns related to anthocyanin biosynthesis in the stem epidermis. The bar and line graphs represent the qRT-PCR and RNA-seq data, respectively.
Figure 7. (A) qRT-PCR and RNA-seq data on gene expression patterns related to anthocyanin biosynthesis in the leaf epidermis. (B) qRT-PCR and RNA-seq data on gene expression patterns related to anthocyanin biosynthesis in the stem epidermis. The bar and line graphs represent the qRT-PCR and RNA-seq data, respectively.
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Figure 8. Validation and analysis of the qRT-PCR results for the BrPAP2 gene.
Figure 8. Validation and analysis of the qRT-PCR results for the BrPAP2 gene.
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Table 1. Genetic analysis of purple-leaf traits in purple pak-choi ‘1395’ × zicaitai ‘1799’.
Table 1. Genetic analysis of purple-leaf traits in purple pak-choi ‘1395’ × zicaitai ‘1799’.
MaterialQuantity of PurpleQuantity of GreenTotalExpected
Ratio		χ2 Test
(Chi−Squared Test)
‘1395’10
‘1799’ 10
F1-310
F2-3157572083:1χ2 = 0.024 < χ20.05,1 = 3.841
Table 2. Genetic analysis of purple-stem traits in purple pak-choi ‘1395’ × zicaitai ‘1799’.
Table 2. Genetic analysis of purple-stem traits in purple pak-choi ‘1395’ × zicaitai ‘1799’.
MaterialPurple
Stem		Green
Stem		Purple
Dwarf Stem		Green
Dwarf Stem		Total
‘1395’ 10 10
‘1799’10 10
F1-3 10 10
F2-3020864144208
Table 3. Correlation of anthocyanin metabolism with MYB-TFs.
Table 3. Correlation of anthocyanin metabolism with MYB-TFs.
Gene IDArabidopsis HomologGene Family
BraA05g000480.3CAtMYB12R2R3-MYB
BraA03g024500.3CAtMYB12R2R3-MYB
BraA06g034790.3CAtMYB111R2R3-MYB
BraA09g004490.3CAtMYB111R2R3-MYB
BraA07g009880.3CAtPAP1R2R3-MYB
BraA07g032100.3CAtPAP2R2R3-MYB
BraA02g017040.3CAtPAP2R2R3-MYB
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BraA04g032060.3CAtCPCR3-MYB
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MDPI and ACS Style

Zhu, Y.; Wang, X.; Tu, X.; Zhang, S.; Zhang, S.; Zhang, H.; Sun, R.; Xu, X.; Gao, X.; Li, G.; et al. Expression Pattern Analyzation and Regulation Genes Identification on the Purple Phenotype in the Different Tissues of the Purple Pak-choi and Zicaitai. Horticulturae 2025, 11, 109. https://doi.org/10.3390/horticulturae11020109

AMA Style

Zhu Y, Wang X, Tu X, Zhang S, Zhang S, Zhang H, Sun R, Xu X, Gao X, Li G, et al. Expression Pattern Analyzation and Regulation Genes Identification on the Purple Phenotype in the Different Tissues of the Purple Pak-choi and Zicaitai. Horticulturae. 2025; 11(2):109. https://doi.org/10.3390/horticulturae11020109

Chicago/Turabian Style

Zhu, Yating, Xia Wang, Xiuping Tu, Shujiang Zhang, Shifan Zhang, Hui Zhang, Rifei Sun, Xiuxiu Xu, Xinyu Gao, Guoliang Li, and et al. 2025. "Expression Pattern Analyzation and Regulation Genes Identification on the Purple Phenotype in the Different Tissues of the Purple Pak-choi and Zicaitai" Horticulturae 11, no. 2: 109. https://doi.org/10.3390/horticulturae11020109

APA Style

Zhu, Y., Wang, X., Tu, X., Zhang, S., Zhang, S., Zhang, H., Sun, R., Xu, X., Gao, X., Li, G., & Li, F. (2025). Expression Pattern Analyzation and Regulation Genes Identification on the Purple Phenotype in the Different Tissues of the Purple Pak-choi and Zicaitai. Horticulturae, 11(2), 109. https://doi.org/10.3390/horticulturae11020109

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