Identification of a Leaf Cuticular Wax Biosynthesis Gene BrCER2 in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

Glossy appearance is a critical trait that affects the appearance quality and marketability of leafy vegetables, including Chinese cabbage. The glossy trait is primarily associated with cuticular wax. Although several genes involved in cuticular wax biosynthesis have been characterized in Chinese cabbage, the regulatory relationships among them remain unclear. In this study, we identified a glossy mutant, glossy leaf4 (gl4), and cuticular wax crystals in the gl4 mutant were obviously reduced. Genetic analysis indicated that the glossy phenotype in the gl4 mutant appears to be controlled by a single recessive gene. Using a bulked segregant analysis coupled with next-generation sequencing (BSA-seq) and map-based cloning methods, the AtCER2 homologous gene BrCER2 was identified as the candidate gene. BrCER2 was expressed in various tissues, and BrCER2-GFP was localized in the endoplasmic reticulum (ER). Furthermore, BrCER2 could interact with BrKCS6 in the ER, and the expression levels of some wax biosynthesis-related genes were decreased in the gl4 mutant. Our overall results provide insights about the role of BrCER2 in wax biosynthesis through ER localization and interaction with BrKCS6 in Chinese cabbage.

1. Introduction

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the cruciferous vegetable crops with the largest cultivation area and consumption in China. Plant cuticular wax is a hydrophobic barrier that appears as a glaucous frost-like substance on the surface of plant tissues, known as wax powder in cruciferous vegetable crops [1]. Cuticular wax not only enhances the plant’s resistance to stress, such as drought, high temperature, and diseases and pests, but also determines the appearance quality and commercial value [2,3,4]. For cabbage-type vegetable crops with leaves or flowering stalks as their product organs, those without wax powder have a fresh appearance and good commercial traits, which are more favored by consumers. Cuticular wax is a mixture composed of various substances, and its inheritance is controlled by multiple genes [3]. Research about cuticular wax formation has been extensively investigated in Arabidopsis and other model plants [5]. However, the corresponding research about cuticular wax remains relatively limited in Chinese cabbage.

The epidermal wax of plants is primarily composed of very-long-chain fatty acids (VLCFAs) and their derivatives, cyclic compounds, and certain secondary metabolites. Its biosynthetic pathway can be divided into three major steps: de novo fatty acid synthesis, VLCFA elongation, and wax derivative formation. In Arabidopsis, de novo fatty acid synthesis is catalyzed by the plastid fatty acid synthase complex (FAS), catalyzing the production of C16 and C18 fatty acids. CER8/LACS1 further converts them into C16 and C18 fatty acyl-CoA esters [6,7,8]. In maize, ZmENR1, encoding a plastid-localized enoyl-acyl carrier protein (ACP) reductase, is involved in the de novo fatty acid biosynthesis [9]. The VLCFAs (up to C28) are subsequently synthesized by the fatty acid elongase (FAE) complex, which comprises four core enzymatic components: β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD), and enoyl-CoA reductase (ECR) [10]. For chain elongation beyond C28, CER2 and its homologs are essential for coordinating the extension process [11,12]. Furthermore, the CER2-KCS6 module can collectively regulate pollen hydration and male sterility in rice and Arabidopsis [13,14]. Then, VLCFAs are subsequently channeled into two major pathways of the decarbonylation pathway and the acyl reduction pathway to generate additional wax components [3,15]. In Arabidopsis, CER1 is a key enzyme in the decarbonylation pathway and interacts with CER3 and CYTB5s. CER1 and CER3 serve as core components in the biosynthesis of very-long-chain alkanes, while CYTB5s is the electron donor that is necessary for the enzymatic activity of CER1 [16,17]. The acyl reduction pathway involves WSD1, responsible for wax ester biosynthesis, and CER4, essential for primary alcohol formation [18,19]. A recent study reported that SOH1 encodes a putative aldehyde reductase and cooperates with CER3 to establish a novel two-step reductive synthesis pathway for primary alcohol synthesis [20].

In cruciferous vegetable crops, several cuticular wax-related genes have been cloned using the glossy leaf materials; most of the cloned genes encode key enzymes in the cuticular wax biosynthesis pathway. In Brassica oleracea, the candidate genes of Cgl1 and Cgl2 are the homologs of Arabidopsis CER1 and CER4, respectively, and the corresponding mutants exhibit glossy and brilliant green phenotypes [21,22,23]. In Chinese cabbage, BrWAX2 and BrWDM1, encoding the Arabidopsis CER1 and CER4 homologous proteins, respectively, were also cloned by utilizing glossy mutants [24,25]. To date, CER2/CER2-LIKE genes and a few FAE complex components have been identified in cruciferous vegetable crops. In Chinese cabbage and broccoli, the candidate genes of BrWAX1/BrCER2 and BoGL5 were identified as the Arabidopsis CER2 homologous gene; the coding sequence mutation of BrCER2 and BoGL5 was responsible for the glossy leaf and stem phenotypes [26,27,28]. In addition, BrWAX3 and BrKCS6 encode β-ketoacyl-CoA synthases (KCSs) of the FAE complex, which participate in VLCFA biosynthesis [29,30]. In Arabidopsis, the CER2/CER2-like proteins can interact with BrKCS6 to regulate pollen hydration, and AtKCS3 functions as a negative regulator in stem wax biosynthesis by interacting with AtKCS6 and reducing its activity [31]. However, knowledge about the molecular relationships between these wax biosynthesis regulators is still limited in Chinese cabbage.

In this study, we identified and characterized a glossy leaf mutant gl4 in Chinese cabbage. Through BSA-seq and map-based cloning, we identified BraA01g015290.3C (BrCER2) as the candidate gene underlying the phenotype and analyzed its function through expression, localization, and interaction assays.

2. Results

2.1. The gl4 Mutant Shows a Glossy Phenotype

To identify genetic factors regulating leaf glossiness in Chinese cabbage, we identified a glossy mutant named glossy leaf 4 (gl4) from the inbred line Br74. Compared with waxy wild-type plants at the bolting and blooming stage, the gl4 mutant showed a glossy phenotype in many aerial organs, such as the leaves, stems, and flower buds (Figure 1).

To investigate the cause of the glossy phenotype in the gl4 mutant, we observed the microscopic structure of cuticular wax in leaves and stems by a scanning electron microscope (SEM). The SEM analysis showed that the wild-type had many wax crystals on the surface of the leaf and stem, and wax crystals were mainly columnar in the wild-type (Figure 2A,B). But, gl4 had fewer wax crystals on the surface of the leaf and stem (Figure 2A,B). These results suggest that the reduction in cuticular wax is associated with the glossy phenotype of the gl4 mutant.

2.2. The Cuticle Permeability of the gl4 Mutant Is Decreased

The changes in cuticle composition are closely related to leaf permeability. To test the difference between wild-type and gl4 in leaf permeability, we performed the toluidine blue (TB) staining assays. The leaf of the wild-type was barely stained, but the leaf of the gl4 mutant was mildly stained (Figure 3A). Then, we also performed the chlorophyll extraction rate assay and water loss rate analysis. The results showed that the chlorophyll extraction rate of the gl4 mutant was higher than that of the wild-type at 6 h, and the water loss rate of the wild-type was similar to that of the gl4 mutant (Figure 3B,C, Table S1). These results indicated that the cuticle permeability of the gl4 mutant is reduced.

2.3. Fine Mapping of the Candidate Gene

To isolate the causal gene in the gl4 mutant, we first performed genetic analysis. We crossed the gl4 mutant with the wild-type and generated an F₂ population. The F₁ plants were waxy and similar to the wild-type, and the ratio of the 162 waxy plants and 52 glossy plants was close to 3:1 (χ² = 0.056 < χ²_{[df = 1, p = 0.05]} = 3.84, p > 0.05) in the F₂ population. These results indicate that the gl4 phenotype was controlled by a single recessive gene.

To clone the mutant gene in the gl4 mutant, we performed the BSA-seq assay. Firstly, we generated an F₂ population from a cross between the gl4 mutant and the non-heading Chinese cabbage inbred line Bc17 with a waxy leaf. In the F₂ population, we selected 25 F₂ plants with the gl4 phenotype (glossy leaf) and 25 F₂ plants with the Bc17 phenotype (waxy leaf) to construct a gl4 phenotype mixed pool and a Bc17 phenotype mixed pool, respectively. Then, the gl4 phenotype pool, the Bc17 phenotype pool, the gl4 mutant, and Bc17 underwent whole-genome re-sequencing. The index of SNP/InDel and ΔSNP/InDel-index was calculated in Bc17, the gl4 mutant, the gl4 phenotype pool, and the Bc17 phenotype pool. From BSA-seq analysis, the ΔSNP-index of the 4.43 Mb–12.54 Mb region on chromosome A01 was significantly higher than the blue threshold line (Figure 4A). Therefore, the mutant gene was located in the 4.43 Mb–12.54 Mb region on chromosome A01. To further narrow the candidate region, we used 57 glossy plants for fine mapping the candidate gene. Finally, the mutant gene was limited to a 1.07 Mb region between markers A1-72 and A1-81 on chromosome A01 (Figure 4B). In the candidate region, BraA01g015290.3C was homologous to AtCER2 in Arabidopsis thaliana. Compared to the wild-type, the first exon of BraA01g015290.3C in the gl4 mutant contained a 40 bp deletion and a 130 bp insertion, leading to a frame shift and premature termination, and the first intron of BraA01g015290.3C in the gl4 mutant had a 1 bp deletion, resulting in no change in coding sequence (Figure 4C and Figure S1). A 40 bp deletion and a 130 bp insertion were further confirmed by sequencing and PCR analysis (Figure 4D and Figure S1). Therefore, BraA01g015290.3C was selected as the candidate gene and named BrCER2.

2.4. Expression Pattern and Subcellular Localization of BrCER2

To investigate the biological function of BrCER2, we performed quantitative real-time PCR (qRT-PCR) analysis using various tissues. The qRT-PCR analysis result showed that BrCER2 was expressed in many tissues and was highly expressed in leaves (Figure 5A). To determine the subcellular localization of the BrCER2 protein, we constructed a BrCER2-GFP fusion protein and transformed it into N. benthamiana leaves. Transient expression of the BrCER2-GFP fusion protein assay show that BrCER2-GFP is localized to the ER (Figure 5B).

2.5. Expression of Wax-Related Genes Is Down-Regulated in the gl4 Mutant

To analyze whether the BrCER2 mutation affects transcription levels of wax-related genes, we examined the expression level of various wax-related genes in the leaves of the gl4 mutant and wild-type plants at the bolting and blooming stages by qRT-PCR analysis. The expression level of wax biosynthesis-related genes differed between the gl4 mutant and wild-type (Figure 6). Compared with the wild-type, the expression levels of VLCFA biosynthesis genes, including BrKCS6, BrKCR1, and BrECR, were significantly reduced in the gl4 mutant (Figure 6). In addition, the transcript levels of the alkane-forming pathway and alcohol-forming pathway genes, including BrCER1, BrWSD1, and BrCER4, were reduced in the gl4 mutant (Figure 6). These results suggest that the BrCER2 mutation might influence the normal expression of wax-related genes.

2.6. BrCER2 Interacts with BrKCS6

CER2 can interact with KCS6 in rice and Arabidopsis, and the BrKCS6 mutation conferred a glossy phenotype in Chinese cabbage [13,14,29]. Therefore, we speculated that BrCER2 may interact with BrKCS6 to regulate the glossy phenotype in Chinese cabbage. Then, we performed a bimolecular fluorescence complementation (BiFC) assay to test the interaction between BrCER2 and BrKCS6 in N. benthamiana leaf cells. BrCER2 was fused with the C terminus of the yellow fluorescent protein (BrCER2-cYFP), and BrKCS6 was fused with the N terminus of the yellow fluorescent protein (BrKCS6-nYFP). A strong yellow fluorescent protein signal was observed in N. benthamiana leaf cells when BrCER2-cYFP was co-expressed with BrKCS6-nYFP, but no fluorescent protein signal was observed in other co-expressed combinations, including the BrCER2-cYFP/nYFP empty vector, BrKCS6-nYFP/cYFP empty vector, and cYFP/nYFP empty vectors (Figure 7A,C). And, the interaction position of BrCER2 and BrKCS6 was localized to the ER (Figure 7A). We also found that BrCER2 and BrKCS6 can interact with themselves, respectively, and BrKCS6 also functions in the ER (Figure 7B,D and Figure S2). These results show that BrCER2 can interact with BrKCS6 in Chinese cabbage.

3. Discussion

Leaves are the product organs in Chinese cabbage, and the glossy green phenotype of leaves is a crucial aspect of appearance and commercial quality. But, knowledge about gene cloning and the molecular mechanism of wax regulators is quite limited in Chinese cabbage. In this study, we identified a gl4 mutant with a glossy leaf in Chinese cabbage. The reduction in cuticular wax crystals resulted in a glossy phenotype in the gl4 mutant. The toluidine blue staining assay and chlorophyll leaching assay indicated that the leaf permeability increased less in the gl4 mutant. Based on BSA-seq analysis and the map-based cloning assay, BraA01g015290.3C was found to be the homolog of AtCER2 and identified as the candidate gene. Furthermore, BrCER2 was localized to the ER and interacted with BrKCS6. Our findings enhanced the understanding of BrCER2 molecular function in regulating the glossy leaf trait in Chinese cabbage.

AtCER2 belongs to the BAHD acyltransferase family and is involved in VLCFA elongation beyond C28 [12]. BrCER2 shared 74.1% identity with AtCER2, and BrCER2 may have a similar function as AtCER2 in VLCFA elongation. BrCER2 is responsible for C28 fatty acid elongation [27]. Furthermore, BrCER2 was localized in the ER (Figure 5B), which is consistent with the action site of the FAE complex in VLCFA elongation. In Chinese cabbage, the gl4 mutant, HN19-G material, and 08A235-2 material had a 40 bp deletion and a 130 bp insertion in the first exon of BrCER2 [27,28]. The BrCER2 of HN19-G material was inserted by a partial LINE-1 retrotransposon (BrLINE1-RUP) sequence [27]. The formation mechanism of the first exon sequence variation in the BrCER2 of the gl4 mutant, HN19-G material, and 08A235-2 material may be similar in Chinese cabbage. But, the gl4 mutant is not the same material as HN19-G and 08A235-2 because the gl4 mutant has a unique 1 bp deletion in the intron (Figure 4C). In addition, the function of BrCER2 in the glossy phenotype regulation needs transgenic verification in Chinese cabbage. Some recent studies have reported that mutations in BrMYB31, BrBCAT1, and BrKCS6 are responsible for the glossy phenotype and that these mutants exhibit a few surface wax crystals in the leaf along with significantly increased cuticle permeability in Chinese cabbage [2,29,32]. But, we found that the cuticle permeability in the gl4 mutant increased less compared with that in the wild-type using the TB staining assay, chlorophyll extraction rate assay, and water loss rate assay (Figure 3). So, the gl4 mutant is a good genetic resource for quality breeding in Chinese cabbage. Furthermore, the coding sequence mutation of BrCER2 could be used for designing the molecular marker for marker-assisted selection (MAS) breeding in Chinese cabbage.

CER2 and CER2-LIKE proteins have been characterized in some plant species, including Arabidopsis, rice, broccoli, and Chinese cabbage [26,27,33,34,35]. However, CER2 proteins may have divergence of gene function and expression level in the various tissues of different plant species. The levels of C30 primary alcohol and C29 aldehydes were dramatically decreased in the cer2 mutant in Arabidopsis, but the level of C30 primary alcohols showed no significant difference in the BrCER2 mutant in Chinese cabbage, and the level of C29 aldehydes increased in the BoCER2 mutant in broccoli [26,27,33]. So, the function of CER2 may have diverged in Arabidopsis, Chinese cabbage, and broccoli. In Arabidopsis, CER2 was only expressed in the epidermis of young siliques and stems, resulting in the cer2 mutant with glossy stems, and their phenotype and wax components had no difference in leaves [33,36]. And, the gl4 mutant, HN19-G material, and 08A235-2 materials had glossy traits in various tissues (Figure 1), including leaves, stems, and flowers [27,28]. Similarly, BoGL5/BoCER2 was also highly expressed in leaves, and the corresponding mutant also showed a glossy appearance in various tissues [26]. Therefore, the different phenotypes between Chinese cabbage and Arabidopsis in leaves may be due to the different expression pattern of BrCER2 in the leaves (Figure 5A). In addition, the total amounts of wax in the leaf were far lower than those in the stem in Arabidopsis, and the different wax amounts between leaf and stem may contribute to phenotypic differences [31]. For example, the AtKCS6 mutant and AtKCS3 overexpression lines obviously reduced total wax amounts in the leaf and stem, but only the stems exhibited a glossy phenotype [31]. SEM analysis showed that the gl4 mutant had few wax crystals in leaves and stems compared to the wild-type (Figure 2A,B). Therefore, it is needed to further investigate the regulatory mechanism of CER2 at the transcriptional level among Arabidopsis and Chinese cabbage in future studies.

CER2 and its homologs and FAE complex play important roles in VLCFA elongation, and CER2/CER2-LIKE proteins could interact with different FAE complex components [37]. CER2 could interact with the FAE complex component KCS6 in Arabidopsis and rice [13,14,34]. CER2/CER2-LIKE proteins could influence KCS6 activity in the VLCFA elongation pathway [11,38]. The CER2-KCS6 module plays important roles in different growth and development processes. The CER2-KCS6 module could affect pollen hydration in Arabidopsis, and it also influenced leaf cuticular wax synthesis and male sterility in rice [13,14,34]. Recently, AtCER19 encodes acetyl-CoA carboxylase1 and catalyzes the synthesis of malonyl-CoA. And, AtCER19 interacts with AtCER2 and affects AtCER2 activity in VLCFA elongation. AtCER2 may act as an adaptor to mediate the formation of the AtCER19-AtCER2-AtKCS6 complex [39]. However, the relationship between CER2 and KCS6 is unclear in Brassica vegetable crops, including Chinese cabbage. In Chinese cabbage, BrKCS6, encoding 3-ketoacyl-CoA synthases, was identified as the candidate gene of wdm9 and wdm10, and the two mutants had a glossy appearance in various tissues, including leaves, stems, and siliques [29]. The glossy phenotype of the BrKCS6 mutant was similar to that of the gl4 mutant. The BiFC assay showed that a strong fluorescent signal was observed when BrCER2-cYFP was co-expressed with BrKCS6-nYFP in N. benthamiana leaf cells (Figure 7A). Thus, this interaction was conserved in Chinese cabbage. And, the expression levels of wax-related genes were changed in leaves of the gl4 mutant (Figure 6). BrKCS6, BrKCR1, and BrECR, involving VLCFA biosynthesis genes, were significantly reduced; BrCER1, BrWSD1, and BrCER4, involving alkane-forming and alcohol-forming pathways, were also reduced in the gl4 mutant (Figure 6). BrKCS6 is the FAE complex component, and its mutant also affects the transcriptional level of wax-related genes [29]. Therefore, the role of BrCER2 in VLCFA and wax biosynthesis further expanded our knowledge about CER2 proteins.

In conclusion, we identified a glossy leaf mutant gl4 and revealed that BraA01g015290.3C (BrCER2) was identified as the candidate gene. BrCER2 encoded BAHD acyltransferase and interacted with BrKCS6 in the ER. The present findings provide a new insight for breeding bright leaf varieties and a better understanding of the wax biosynthesis pathway in Chinese cabbage. In addition, CER2 and CER2-LIKE proteins had an important role in the development of plant pollen. AtCER2 and AtCER2L2 were likely involved in pollen hydration, and the VLCFAs contents of the cer2 cer2L2 double mutant were decreased in the pollen coat, resulting in defective hydration and male sterility [14]. In rice, OsCER26L was identified as an HMS1-interacting protein and played an important role in humidity-sensitive genic male sterility [13]. And, BrCER2 was also expressed in the flower (Figure 5A). Thus, it is necessary to clarify the function of BrCER2 in anther and pollen development in Chinese cabbage in future studies.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The wild-type Br74 was a Chinese cabbage inbred line from the Kangaibaicai variety, and the gl4 mutant was identified from Br74. The gl4 mutant had a glossy phenotype, and wild-type plants had a waxy appearance in the bolting and blooming stages of the leaf, stem, and pistil. For genetic analysis, Br74 and gl4 mutant were used to generate F₁ plants and an F₂ population. For BSA-seq analysis and gene mapping, the non-heading Chinese cabbage lines Bc17 and the gl4 mutant were used as parents to generate the F₂ population. The germinated seeds were transferred to a 4 °C refrigerator for 20 days of vernalization. The vernalized seeds were cultivated in a growth chamber maintained at 70% relative humidity, a photosynthetic photon flux density (PPFD) of 600 μmol m⁻² s⁻¹, a 16 h light/8 h dark photoperiod, and a constant temperature of 24 °C. After 25 days, plant materials were cultured in the plastic house at Zhejiang A&F University.

4.2. Scanning Electron Microscopy Analysis

The fresh leaves and stems of the wild-type and the gl4 mutant were collected at the bolting stage. The leaves and stems were cut into 0.5 cm squares and dried in a vacuum freeze-drier for 36 h. The dehydrated samples were plated with gold using the ion sputter coater (SBC-12, KYKY, Beijing, China) for 40 s. And, the morphology of cuticular wax crystals was observed by the scanning electron microscope (TM4000 plus, Hitachi, Tokyo, Japan) at 15 kV.

4.3. Toluidine Blue (TB) Test

The toluidine blue test was performed using leaves at the bolting stage as described previously [40]. The fresh leaves of wild-type and gl4 mutant plants were collected at the bolting stage. The fresh leaves were incubated in 0.05% (w/v) toluidine blue for 20 min and then washed with water to remove the toluidine blue from the leaf surface.

4.4. Chlorophyll Leaching Assay and Measurement of Water Loss

Chlorophyll leaching assay was performed using the leaves of the wild-type and gl4 mutant at the bolting stage. Approximately 2 g of leaves was immersed in 30 mL of 80% ethanol at room temperature. The 3 mL aliquot of each sample was taken every hour. And, the chlorophyll amount in the aliquot was determined by measuring absorbance at 647 nm and 664 nm wavelength using a UV/VIS spectrophotometer (UV-5500, Shanghai METASH instruments company, Shanghai, China). The concentration and rate of total chlorophyll extracted were calculated as described previously [40]. The concentration of total chlorophyll was calculated using the following equation: total micromoles chlorophyll = 7.93 (A664)  +  19.53 (A647). The rate of total chlorophyll extracted was calculated by the percentage of the different times of chlorophyll over the total chlorophyll extracted after 24 h.

The leaves at the bolting stage were used to conduct water loss rate analysis. The leaves were dried at room temperature, weighed every one hour using a microbalance, and then dried at 65 °C until the dry weight remained constant. Total water content was calculated by the fresh weight minus the dry weight. The rate of water loss was calculated as the percentage of water loss over the total water.

4.5. BSA-Seq Analysis

For BSA-seq analysis, the F₂ population was generated by a cross between the gl4 mutant and the non-heading Chinese cabbage inbred line Bc17 with a waxy leaf. The waxy phenotypes of Bc17, gl4 mutant, F₁ plants, and F₂ population were observed. Equal amounts of DNA from 25 F₂ plants with the glossy leaf phenotype and 25 F₂ plants with the waxy leaf phenotype were pooled to construct a glossy mixed pool and a waxy mixed pool, respectively. DNA from the two pools, Bc17 and the gl4 mutant, was sequenced on an Illumina Hiseq^TM PE150 platform. These samples used the B. rapa genome v3.0 as the reference genome. The sequencing depth and coverage of four samples in the BSA-seq assay are listed in Table S2. A BWA (Burrows–Wheeler Aligner) was used to align the clean reads of each sample against the reference genome. The SNP index and INDEL index were calculated at each position of the glossy mixed pool and the waxy mixed pool according to the read depth information for homozygous SNPs/InDels. The ΔSNP/InDel-index of each SNP/InDel position was calculated by subtracting the SNP/InDel-index of the glossy mixed pool from the SNP/InDel-index of the waxy mixed pool. SNP/InDel and ΔSNP/InDel indices were analyzed following a previously described method [41]. The BSA-seq analysis was performed by the Novogene company (Peking, China).

4.6. Map-Based Cloning

For genetic analysis, an F₂ population was generated from a cross between the gl4 mutant and the wild-type Br74, and the ratio of the waxy and glossy phenotypes was obtained. For map-based cloning, we first generated an F₂ population from a cross between the gl4 mutant and the non-heading Chinese cabbage inbred line Bc17 with a waxy leaf, and ten F₂ plants with the recessive glossy leaf phenotype were used for preliminary mapping. Then, 57 glossy leaf plants from the F₂ population were selected for fine mapping. InDel/SSR markers were designed using Primer Premier 5.0 software according to the genomes of Chiifu (B. rapa cv. Chiifu genome v3.0, Chinese cabbage) and pak choi (B. rapa cv. Pak choi genome v1.0, non-heading Chinese cabbage) posted on the BRAD website. The polymorphism screening of InDel primers in the gl4 mutant, Bc17, and the waxy and glossy pools was performed using polyacrylamide gel electrophoresis. The screened polymorphic InDel/SSR markers were used to fine-map the mutant gene. Molecular markers for fine mapping are listed in Table S3.

4.7. DNA, RNA Extraction, and qRT-PCR Analysis

Fresh leaves were harvested for DNA isolation using the cetyltrimethylammonium bromide (CTAB) method. For the expression pattern assay, the total RNA from fresh leaves, stems, flowers, siliques, and buds was extracted using the RNA prep Pure Plant Kit (TIANGEN, Peking, China). For the wax-related gene expression assay, the fresh leaves from the gl4 mutant and Br74 were harvested and isolated from total RNA using the RNA prep Pure Plant Kit. A total of 1 μg of total RNA was used to synthesize first-strand complementary DNA (cDNA) using the Takara Prime Script 1st Strand cDNA Synthesis kit. The BrACTIN gene was used as an internal control, and the primers of wax biosynthesis-related genes were used as described previously [32]. Quantitative RT-PCR assays were performed on a qTOWER3/G real-time PCR machine (Jena, Germany) using a SYBR Premix Ex Taq^TM kit (Takara, Peking, China). The 2^-ΔΔCt method was used to calculate relative expression levels. The primers for the qRT-PCR assay are listed in Table S3.

4.8. Subcellular Localization of BrCER2 Protein

For the subcellular localization of BrCER2, the C-terminus of BrCER2 was fused to GFP protein in the pCAMBIA1305.1 vector. The ER-rk CD3-959 fusing to mCherry protein was used as the ER localization marker. These constructs were transformed into the Agrobacterium strain EHA105. Then, EHA105 containing constructs were transformed into tobacco mesophyll cells, and the tobacco was incubated for 2 days at 28 °C. Fluorescence signals of GFP and mCherry protein were observed on a confocal laser scanning microscope (Olympus FV3000, Tokyo, Japan).

4.9. Bimolecular Fluorescence Complementation Assays

For BiFC assays, the full-length coding sequence of BrCER2 and BrKCS6 was inserted into p2YN and p2YC vectors, respectively. BiFC assays were conducted according to a previously described method [42]. These plasmids were transformed into the Agrobacterium strain EHA105, and different combinations of these constructs were co-transfected into tobacco mesophyll cells. And, the tobacco was incubated for 2 days at 28 °C. The ER-rk CD3-959 fusing to mCherry protein was used as the ER localization marker. Fluorescence signals of YFP and mCherry proteins were detected using the confocal laser scanning microscope (Olympus FV3000, Tokyo, Japan). The fluorescence intensity of the YFP protein assay was measured using ImageJ 1.53e software. Primers used for BiFC assays are listed in Table S3.

4.10. Statistical Analysis

Statistical analysis was calculated using IBM SPSS software 19.0. Significant differences were analyzed through Student’s t-test (* p < 0.05; ** p < 0.01).

5. Conclusions

In this study, we identified BrCER2 and provided new insights about the function of BrCER2 in glossy leaf regulation in Chinese cabbage. The glossy appearance of the gl4 mutant is caused by the reduction in cuticular wax. Using BSA-seq analysis combined with a map-based cloning approach, BrCER2 was identified as the candidate gene. BrCER2 was expressed in various tissues, especially in leaves. The BrCER2 mutation resulted in reducing the expression levels of some wax-related genes, including BrKCS6, BrCER1, and BrWSD1. Furthermore, BrCER2 was localized in the ER and could interact with BrKCS6 and itself. Our findings advance understandings of the function of BrCER2 in the wax biosynthesis network in Chinese cabbage.