Identification of BoFAR3a Reveals the Genetic Basis of a Glossy Green Trait in Broccoli

Abstract

Mutants with a bright green appearance due to wax synthesis or deposition defects have been reported in various plants such as Arabidopsis thaliana, corn, and rice, but they are relatively rare in broccoli (a brassicaceae crop). Here, we describe SY03, a natural mutant of broccoli with a glossy green phenotype owing to epidermal wax deficiency. Genetic analysis indicated that the leaf luster trait of SY03 was controlled by a single recessive gene. By using the F₂ generation and combining bulked segregant analysis and molecular marker techniques, the candidate gene BoFAR3a, homologous to the Arabidopsis FAR gene, was identified within a 96.678 kb interval of chromosome C01. The A→G point mutation in exon 1 of the BoFAR3a coding sequence substitutes the canonical ATG start codon with GTG, which is predicted to abrogate or severely reduce translation initiation. RT-qPCR indicated that the expression levels of BoFAR3a were significantly decreased in the leaves of the glossy green phenotype mutant. Heterologous expression of BoFAR3a in A. thaliana restored the phenotype of A. thaliana mutant FAR3. The discovery of BoFAR3a is of great significance for breeding lustrous and commercially appealing broccoli varieties. This study systematically analyzed the molecular basis of the lustrous green phenotype in broccoli, providing new insights into the epidermal waxy regulatory network of cruciferous crops. In the future, the wax synthesis pathway can be precisely improved through gene editing technology, achieving a coordinated enhancement of the appearance quality and stress resistance of broccoli.

1. Introduction

Wax powder is a complex mixture of organic substances secreted by the plant epidermis. Its main components include long-chain fatty acids (C20–C40), their derivatives (such as alkanes, ketones, and alcohols), and terpenoids [1]. The characteristics and composition of plant surface wax powder also differ depending on the species and growing environment. Wax powder usually makes the plant surface grayish-white, but in mutants lacking wax powder, it can appear shiny green. Wax powder provides physiological protection, facilitates environmental adaptation, and affects ecological interactions, including water regulation and drought resistance. The wax layer protects against visible and UV light, prevents diseases by serving a barrier to pests, promotes organ development and morphological maintenance, and confers hydrophobic and anti-adhesive properties [2,3,4,5,6].

In Arabidopsis thaliana and other plants, CER and gl mutants have been used to identify several genes associated with wax synthesis, including those encoding wax synthesis-related enzymes (CER4 [7], CER6 [8], CER10 [9], FATB [10], and GL8 [11]), wax transporters (CER5 [12] and WBC11 [13]), and transcription factors (SHN1 [14], MYB30 [15], MYB96 [16], and WXP [17]). A. thaliana fatty acyl-CoA reductase (FAR), encoded by CER4, plays a key role in plant epidermal wax synthesis by catalyzing the reduction of very long chain fatty acid acyl-CoAs (VLCFA-CoAs) to primary alcohols [7]. This process is one of the core steps in epidermal wax biosynthesis and thus directly affects the barrier function and stress resistance of the epidermis.

In recent years, several genes related to wax synthesis in Brassica crops have been systematically located and functionally analyzed using molecular genetics. Through the genetic map analysis, Liu et al. [18] identified wax synthesis gene Bol013612 in Chinese cabbage, which is directly homologous to CER4 of A. thaliana and also encodes an FAR. By using whole-genome InDel markers, Han et al. [19] fine-mapped the BoGL5 gene, which controls broccoli wax synthesis, to a 94.1 kb region of chromosome C01, where candidate gene BoCER2 was identified through sequence comparison and functional verification. BoCER2 was found to be highly homologous to Arabidopsis CER2. Subsequently, the function of BoGL5 was verified using the CRISPR-Cas9 technology [20]. Introduction of wild-type BoCER2 into the TL28-1 wax-deficient broccoli mutant restored wax powder deposition and normal phenotypes, demonstrating that BoCER2 is the key gene controlling this trait [21]. By using ethyl methanesulfonate chemical mutagenesis, Liu et al. [22] obtained a wax-less mutant of Chinese cabbage and identified BraA09g066480.3C as a key candidate gene responsible for this trait by combining with the MutMap positioning system and KASP molecular marker validation technology. Phylogenetic analysis showed that the product of this gene was homologous to the Arabidopsis CER1 protein (aldehyde decarboxylase), revealing its important role in the epidermal wax alkane synthesis pathway. Song et al. [23] described Chinese cabbage glossy allelic mutants wdm4 and wdm8. By analyzing the phenotypic variation characteristics of the hybrid offspring between mutants and using a localization cloning strategy, the authors identified BraA01g015290.3C as the target gene, which was homologous to Arabidopsis CER2, known to facilitate acyl chain elongation during cuticular wax production.

2. Results

2.1. Phenotypic Characteristics and Genetic Analysis of the SY03 Mutant

SY03 is a natural broccoli mutant with a deletion in epicuticular wax. Compared with the T104-1 phenotype (Figure 1a,c), all plant organs of SY03, including the leaves (Figure 1b), flower buds (Figure 1d), stems (Figure 1f), and pods (Figure 1h), had a glossy green color. Cryo-scanning electron microscopy analysis showed more wax crystals in T104 than in SY03. Wax crystals on T104-1 surfaces had mountain-like protrusions (Figure 2a), whereas almost no wax crystals were observed on SY03 plant parts (Figure 2b).

The F₁ and F₂ generations were obtained using SY03 as the maternal parent and T104-1 as the paternal parent to study the genetic patterns of SY03 wax powder deficiency traits. All F₁ generation plants showed the same grayish-green wax powder phenotype (

Table 1. Genetic analysis of the glossy trait in crosses between T104 and SY03.

Population	Total	Waxy	Glossy	Expected Ratio	X2	X20.05
F1	25	25	0	-	-	-
F2	1270	936	334	3:1	1.143	3.84
BC1	831	422	409	1:1	0.203	3.84

). The separation ratio of the F₂ generation (936 waxy plants and 334 glossy green plants) was 3:1 (X² = 1.143, X²₀._05,1 = 3.84). The separation ratio of the BC₁ generation (422 waxy plants and 409 glossy green plants) was 1:1 (X² = 0.203, X² < ts)._05,1 = 3.84). Therefore, we believe that the glossy green phenotype of SY03 is controlled by a single recessive gene.

2.2. Altered Cuticular Wax Production in Glossy Plants

GC-MS was used to determine the loading amount and chemical composition of the surface wax powder in T104-1 and SY03. As shown in Figure 3, the ketone, fatty acid, aldehyde, and secondary alcohol contents of SY03 and T104-1 were similar. The primary alcohol and wax ester contents in SY03 were significantly lower than those in T104-1. Therefore, the glossy green phenotype of SY03 might be due to the reduced content of primary alcohols and wax esters, which altered the proportion of wax chemical components and thereby significantly decreased the wax powder content.

2.3. Fine Mapping of the Wax Powder Synthesis Gene

Bulked segregant analysis-sequencing (BSA-Seq) method was performed in primary mapping. From the F₂ population, 50 plants with normal wax powder phenotype and 50 plants with glossy green phenotype were selected to construct the wax phenotype bulk (W-bulk) and glossy green phenotype bulk (G-bulk), respectively. Both bulks, along with their parental lines, were subjected to whole-genome re-sequencing. The two progeny bulks yielded approximately 122 million and 123 million raw reads, respectively. The sequencing libraries showed high quality, with an average Q30 base percentage of 92% and an average GC content of 36.5%. After alignment to the HDEM reference genome and stringent filtering, a total of 5,426,787 and 5,433,232 high-confidence single nucleotide polymorphisms (SNPs), as well as 1,014,054 and 1,590,707 insertions/deletions (InDels), were identified in the W-bulk and G-bulk, respectively. By using the sliding window analysis of the absolute Δ(SNP) values, a candidate 0–5.23 Mb interval on chromosome C01 was initially defined at the 99% confidence level (Figure 4A).

To fine-map the interval, 50 SNP markers were developed within the candidate region based on the BSA-Seq results. Of these, 14 markers showed clear polymorphism between the parental lines. These 14 polymorphic markers were then used to genotype 50 plants with normal wax powder and 50 glossy green F₂ plants for linkage analysis. This analysis identified three recombinant plants using marker P1164A14 and five recombinant plants using marker P1164A17, narrowing the candidate interval to a 417 kb region between P1164A14 and P1164A17. Subsequently, we used four markers, P1164A14, P1164A15, P1164A16, and P1164A17, to test 1270 F₂ plants. Eventually, the candidate genes were located within a 96.678 kb region between P1164A15 and P1164A16 (Figure 4B).

According to the gene prediction information in the HDEM, TAIR, and unpublished databases, 14 genes were located within the 96.678 kb mapping region (

Table 2. Predicted genes in the target genomic region.

Gene Name	Start Position	Stop Position	Homologous Gene in A. thaliana	Annotation
BolC01g004910.2J	2803770	2805349	AT4G33880	Transcription factor RSL2
BolC01g004920.2J	2819712	2820032	AT3G58030	Encodes a RING domain E3 ligase.
BolC01g004930.2J	2823899	2825393	AT4G33870	Peroxidase superfamily protein
BolC01g004940.2J	2827891	2828868	AT4G33800	hypothetical protein
BolC01g004950.2J	2843245	2849168	AT4G33790	Encodes an alcohol-forming fatty acyl-CoA reductase
BolC01g004960.2J	2850858	2851929	AT4G33780	ATP phosphoribosyl transferase regulatory subunit
BolC01g004970.2J	2855989	2859925	-	Ras-related protein RABA5a
BolC01g004980.2J	2863606	2866060	AT4G33770	Inositol-tetrakisphosphate 1-kinase 2
BolC01g004990.2J	2866712	2870095	AT4G33760	Aminoacyl tRNA synthetase functions
BolC01g005000.2J	2870903	2872231	AT4G33740	myb-like protein X
BolC01g005010.2J	2874051	2874829	AT4G33730	Member of CAP protein superfamily
BolC01g005020.2J	2881862	2882984	AT4G33720	CAP (Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 protein) superfamily protein
BolC01g005030.2J	2887183	2889808	AT2G14520	CBS domain protein (DUF21)
BolC01g005040.2J	2890173	2891455	AT4G33690	G patch domain protein
BolC01g005050.2J	2892325	2895010	AT4G33680	LL-diaminoheptaneate transaminase

). Among the 14 candidate genes, only BolC01g004950.2J was associated with plant surface wax synthesis. BolC01g004950.2J encodes an acyl-CoA reductase, which is crucial for the synthesis of long-chain fatty acids. This is consistent with the reduced synthesis of alcohols and waxes in the glossy mutant SY03. Sequence analysis indicated that the sequences of BolC01g004950.2J and Arabidopsis FAR3 (CER4) genes have a high degree of homology (89.45%), and the BolC01g004950.2J gene also contains the FAR domain. Therefore, we initially named the candidate gene BolC01g004950.2J as BoFAR3a. We performed RT-qPCR to reveal the expression pattern of BoFAR3a in wild-type T104-1 and mutant SY03 plants. The results showed that the expression of BoFAR3a was significantly downregulated in the stems, leaves, and siliques of T104-1 compared with those of SY03 (Figure 5).

2.4. Functional Characterization of BoFAR3a

To validate the candidate gene, we amplified its full-length coding sequence. A point mutation in the first exon (A→G) was identified, resulting in a substitution of the initiation codon from methionine (ATG) to valine (GTG; Figure 4C). This mutation is predicted to abolish the production of full-length BoFAR3a protein, as efficient translation initiation critically depends on the canonical ATG start codon. Although GTG can function as a non-canonical start site, its markedly reduced translation efficiency would likely lead to a loss of function (Figure 4D). Subsequently, the protein structure was predicted (Figure 4E). The protein encoded by the BoFAR3a gene was found to contain a GDP-mannose dehydration domain and a sterile domain. An amino acid substitution in SY03 occurred in the GDP-mannose dehydration domain.

2.5. Complementation Experiment Using BoFAR3a

Sequence analysis indicated that the sequences of BoFAR3a and Arabidopsis FAR3 (CER4) genes have a high degree of homology (89.45%), and the BoFAR3a gene also contains the FAR domain. To further confirm the relationship between the BoFAR3a gene and the SY03 glossy green phenotype, we ectopically expressed a full-length coding sequence of the BoFAR3a gene in the Arabidopsis cer4 mutant under the control of the 35S promoter. Mutant plants transformed with empty pCambia3301 served as negative controls. We generated a total of 24 transgenic Arabidopsis lines in the FAR3 (cer4) mutant background, comprising 14 BoFAR3a-overexpression and 10 Bofar3a-overexpression lines. Complementation restored the glossy green phenotype and wax loading in the A. thaliana mutant to the wild-type level, whereas plants transformed with the empty vector retained the mutant phenotype. These results indicate that the BoFAR3a gene is an important gene that regulated the synthesis of wax powder in broccoli and that its mutation causes the glossy green phenotype of the SY03 mutant (Figure 6A). We also examined the expression level of BoFAR3a in A. thaliana transgenic plants and found that it was consistent with that in broccoli (Figure 6B).

2.6. Subcellular Localization of BoFAR3a

We designed a construct encoding the BOFAR3a-GFP fusion protein driven by the 35S CaMV promoter and transfected it into A. thaliana leaf epidermal cells to evaluate its subcellular localization. The BOFAR3a-GFP fusion protein co-localized with an endoplasmic reticulum marker, indicating that BoFARa is located in the endoplasmic reticulum, which is consistent with the metabolic function of this protein in the biosynthesis of VLCFA (Figure 7).

3. Discussion

The surface of plants usually has a grayish-white appearance, owing to the presence of wax powder. A. thaliana eceriferum (cer) was the first wax powder-deficient mutant discovered [15]. Numerous glossy green mutants and genes responsible for this phenotype have been identified in many plants, including rice [16], Welsh onion [17], and cucumber [18]. In recent years, genes controlling wax powder synthesis have been reported in plants belonging to the Brassicaceae family. For instance, the BrCER1 gene in Chinese cabbage encodes an aldehyde decarboxylase, and a single base C→T substitution changes the protein structure [19]. The wax synthesis gene BoGL5 of broccoli was identified within a 94.1 kb interval on C01, and the BoCER2 gene homologous to CER2 in A. thaliana was identified as the candidate gene [20,21]. In this study, we used SNP and InDel markers to find a candidate gene responsible for glossy green phenotype within a 96.678 kb interval on chromosome C01 of the naturally mutated broccoli mutant SY03. We identified BolC01g004950.2J as a candidate gene, showed that it was homologous to the Bol013612 gene mutated in cabbage mutant LD10GL, and established that both these genes are homologous to the FAR3 (CER4) gene in A. thaliana [22]. The Bol013612 mutation caused glossy green phenotype owing to the insertion of six nucleotides into the cDNA, which alters protein structure. In eukaryotes, ATG serves as the initiation codon in the vast majority of cases. In the absence of ATG, GTG can alternatively function as a start codon to initiate translation; however, its initiation efficiency is substantially reduced and may even lead to a failure in protein production. In this study, a single-nucleotide substitution (A→G) occurred in BolC01g004950.2J, which changed the initiation codon from methionine (ATG) to valine (GTG). This mutation ultimately resulted in the loss of epicuticular wax, likely because the use of GTG as the start codon reduced transcriptional efficiency and consequently impaired protein production.

Wax synthesis is a complex process involving multiple reactions [24], such as the extension of VLCFA, reduction, decarboxylation, and esterification [25], which are regulated by the coordinated action of multiple proteins encoded by the CER gene family. The A. thaliana CER4 protein is a key fatty acyl-CoA reductase important for the synthesis of plant epidermal waxy substances. CER4 directly converts VLCFA-CoA into primary alcohols without the need for aldehyde intermediates [22]. The primary alcohol content in the stems of A. thaliana CER4 mutants was decreased significantly, but the contents of metabolites, such as aldehydes and alkanes, were increased, confirming the importance of CER4 in the reduction pathway. In addition, CER4 has a high specificity for VLCFA-CoAs with chain lengths of C24 to C26. Heterologous expression of CER4 in yeast generated primary alcohols, further confirming its substrate preference. In this study, GC-MS indicated that the waxy composition of the SY03 mutant underwent significant changes. However, the alkane content was also significantly reduced, which differed from the findings in A. thaliana. We believe that although the BoFAR3a and AtCER4 genes are homologous, there may be other FAR genes with partially redundant functions in cruciferous crops, and their expression or interaction relationships have changed in different contexts, thereby affecting the results of GC-MS. In this experiment, the alkane content decreased, and the trend of change compared with that in the A. thaliana mutant CER4 was questionable. Notably, a recent study identified a two-step reduction pathway for primary alcohol synthesis in Arabidopsis, mediated by CER3 and SOH1. This work revealed that CER3 serves as a metabolic hub, partnering with CER1 for alkane synthesis or with SOH1 for alcohol synthesis [26]. Mutations in the BoFAR3a gene not only affected the synthesis of primary alcohols, but may have also influenced downstream alkanes through metabolic feedback or substrate competition. Alkane synthesis is controlled by homologous genes, such as CER1 and CER3 [27] We hypothesize that in broccoli, the loss of BoFAR3a function may perturb the equilibrium of the CER3-mediated complex, potentially leading to the coordinated downregulation of both derivative pathways. This interesting finding suggests species-specific regulation, warranting future investigation.

Currently, research on the synthesis of plant epidermal wax mainly focuses on the linear wax components, whereas there are relatively few synthetic pathways for branched-chain wax components, such as branched-chain fatty alcohols and branched-chain alkanes. An acyl-activating enzyme 9 (AAE9) involved in the synthesis of branched-chain wax has been cloned and identified in A. thaliana, confirming that it connects two major pathways of branched-chain amino acid metabolism and branched-chain wax synthesis [28]. In this study, GC-MS data indicated that in addition to the decrease in the contents of alcohols and wax esters, the content of alkanes also changed. We hypothesize that there may still be a gene similar to AEE9 that controls the synthesis of wax powder. In the future, we will explore the factors that control alkane content, facilitating future studies of wax synthesis in plants belonging to the Brassicaceae family.

4. Materials and Methods

4.1. Plant Materials

The plant materials used in this study were preserved at the Zhuanghang Experimental Station of the Shanghai Academy of Agricultural Sciences (30°53′ N, 121°22′ E). Mutant SY03 is a natural glossy green phenotype mutant discovered in broccoli high-generation inbred line T104. The F₁ and F₂ populations were constructed using SY03 as the maternal parent (P1) and T104-1 (P2) as the paternal parent. All materials were provided by the Horticulture Research Institute of the Shanghai Academy of Agricultural Sciences (Shanghai, China) and planted at the Zhuangxing Experimental Station of the Shanghai Academy of Agricultural Sciences (Shanghai, China). Phenotypic identification was conducted at the five-leaf and one-heart stages. The chi-squared test was used to determine the separation ratios of the F₂ populations.

4.2. Scanning Electron Microscopy and Gas Chromatography–Mass Spectrometry

Fresh leaves from five-leaf stage plants were fixed overnight in 2% glutaraldehyde, mounted on specimen stubs using a double-sided tape, and coated with gold particles using a SEMPrep2 sputter coater (Nanotech) (Shanghai, China). The phenotype was analyzed using scanning electron microscopy (S-4,800, Hitachi, Japan) with a secondary electron detector at high voltage (10 kV). The gas chromatography–mass spectrometry (GC-MS) analysis of cuticular wax was performed as described by Song et al. [23].

4.3. DNA Isolation and Fine Mapping of the Wax Powder Synthesis Gene

Total genomic DNA was extracted from fresh leaves using the cetyltrimethylammonium bromide (CTAB) method [29], with concentrations adjusted to 30 ng/µL for downstream applications. For bulked segregant analysis-sequencing (BSA-Seq), two DNA pools were constructed: one from 50 F₂ plants displaying the normal wax phenotype and another from 50 F₂ plants exhibiting the glossy green phenotype. These pools were subjected to whole-genome resequencing on an Illumina HiSeq 2500 platform (Illumina, Inc., San Diego, CA, USA). The resulting reads were aligned to the cabbage reference genome (http://brassicadb.org) for variant calling (accessed on 5 June 2024). Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) were identified and compared between the parental lines’ primary mapping.

Based on the BSA-seq results, 50 SNP makers were designed within the candidate region using Primer3 software (v2.5.0). These markers were evenly distributed across the nine chromosomes, with amplicon lengths under 500 bp, GC content between 40 and 50%, and melting temperatures (T_m) of 60–64 °C. The designed SNP markers were first screened against 50 wax-coated and 50 glossy F₂ plants to confirm polymorphism. Subsequently, 1270 F₂ individuals were genotyped with the polymorphic markers. PCR amplification followed by polyacrylamide gel electrophoresis was used to detect polymorphisms and establish genetic linkage [29].

4.4. RNA Isolation, cDNA Synthesis, and RT-qPCR Analysis of the Candidate Gene Expression Levels

Total RNA was extracted from the stems, leaves, and siliques of T104-1 and SY03 parents using the StarSpin Plant RNA Extraction Kit (polysaccharide and polyphenol-rich type) (Shanghai, China). cDNA was synthesized using the Hifair AdvanceFast 1st Strand cDNA Synthesis Kit (No Dye). The protocols were performed in accordance with the manufacturer’s instructions. The quality of the RNA preparations was determined by measuring the OD value and performing agarose gel electrophoresis. After passing the inspection, the RNA preparations were used for the subsequent experiments.

The expression levels of the candidate genes in SY03 and T104 tissues were compared using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Using actin as the internal reference gene and Hieff Unicon^® qPCR TaqMan (Shanghai, China) probe premix as the reactant, the RT-qPCR reactants were prepared and amplified on the CFX 96 Touch real-time fluorescence quantitative PCR detection system. All the samples were analyzed in three biological replicates. The expression levels of candidate genes in the samples were calculated by the 2^−ΔΔCT method.

4.5. Gene Amplification and Sequence Analysis

Reference sequences of the candidate genes were obtained from BRAD (http://brassicadb.cn/) and unpublished databases (accessed on 20 June 2024). Primers were designed based on the reference sequence using Primer 5.0 software. The full-length sequences of the candidate genes were amplified using the genomic DNA of SY03 and T104 as templates. PCR amplification was performed using Q5 high-fidelity DNA polymerase. The Arabidopsis FAR3 (CER4) sequence was downloaded from the Arabidopsis Information Resource (accessed on 1 July 2024)

The amino acid sequences encoded by the candidate genes were analyzed for homology using BLASTP software (BLAST+ 2.17.0). The obtained proteins were compared using DNAMAN6.0 software.

4.6. Plasmid Construction and Arabidopsis Transformation

The BoFAR3a and Bofar3 coding sequences were amplified from 1st Strand cDNA of T104 and SY03, respectively. The purified PCR products were inserted into modified binary vector pBWA(V)BS (reconstructed from pCAMBIA1301) [30] placed downstream of the constitutive 35S promoter. The constructs were transformed into competent Agrobacterium tumefaciens GV3101 and into Arabidopsis cer4 mutants (SALK_000575C) using the floral dip method [31]. Seeds were screened for hygromycin resistance on the Murashige and Skoog medium containing 30 mg/L hygromycin, and its expression level was further confirmed by RT-PCR analysis.

5. Conclusions

In this study, we successfully identified and verified BoFAR3a (corresponding to the genomic annotation BolC01g004950.2J), a key gene regulating cuticular wax biosynthesis in broccoli (Brassica oleracea L. var. italica). This gene is homologous to the fatty acyl-CoA reductase gene FAR3 (CER4) in Arabidopsis. Using map-based cloning, the target trait was mapped to a 96.678 kb interval on chromosome C01. Sequencing revealed an A→G single-nucleotide substitution at the start codon of BoFAR3a in the mutant SY03, which changed the initiating amino acid from methionine (ATG) to valine (GTG). Although this mutation did not substantially alter the predicted protein structure, the low translational efficiency associated with the GTG start codon likely severely impaired protein synthesis, ultimately leading to the wax-deficient phenotype. Heterologous complementation assays demonstrated that BoFAR3a could restore wax deposition in the Arabidopsis cer4 mutant, confirming its biological function. Furthermore, GC-MS analysis indicated that the contents of primary alcohols and wax esters were significantly reduced in the leaves of the mutant.