Quantification and Diversity Analyses of Glucosinolates in 191 Broccoli Genotypes Highlight Valuable Genetic Resources for Molecular Breeding

Abstract

Glucosinolates (GSLs), crucial secondary metabolites in cruciferous vegetables, hydrolyze upon consumption or mechanical damage, forming bioactive compounds with anti-cancer properties, such as glucoraphanin (GRA). Among cruciferous vegetables, broccoli stands out for its high GSL content, which varies significantly among different genotypes. This study aimed to characterize and quantify glucosinolate compounds in broccoli using LC-HRMS2 and UPLC. We identified thirteen GSLs in 191 broccoli genotypes, including seven aliphatic, five indole, and one aromatic glucosinolate. The GSL content in these genotypes ranged from 0.1705 to 5.8174 mg/g (DW). We also explored GSL diversity and content in seven developmental organs, finding high diversity and content in seedling roots and florets. Notably, genotype No. 300 had the highest GSL content (5.8174 mg/g, DW) and GRA (3.1545 mg/g, DW), along with a larger flower bulb diameter (13.4 cm) and a shorter growth stage (11 days), demonstrating its potential for breeding GRA-rich broccoli. To our knowledge, this study encompasses the largest number of broccoli genotypes to date, broadening our understanding of GSLs’ diversity and content in broccoli. These findings may provide valuable resources for future breeding or the commercial cultivation of GRA-rich broccoli.

1. Introduction

Glucosinolates (GSLs) are essential secondary metabolites found in cruciferous plants, with over 156 structures identified [1]. They are categorized into aliphatic, aromatic, and indole groups based on their amino acid precursors [2,3]. Intact GSLs are chemically stable, while isothiocyanates which are formed post-myrosinase degradation display diverse biological activities [4]. In plants, GSLs and myrosinases are stored separately as mechanical injury triggers myrosinase release, hydrolyzing GSLs [5]. Although humans lack myrosinases, residual bacterial colonies in the gastrointestinal tract with myrosinase-like activity can degrade GSLs to form isothiocyanates [6]. Animal studies confirm that GSLs can convert into biologically active products, affecting physiological health [7].

The interest in GSLs and their hydrolysates has spurred the development of extraction and isolation methods. Depending on research needs, GSL analysis methods fall into two categories: intact GSLs and their breakdown products. The chromatography (LC) method is commonly used for GSL quantification [8]. However, the strong polarity of intact GSLs challenges their separation in LC. Many studies have converted intact glucosinolates into desulfoglucosinolates for an easier LC analysis [9,10]. Moreover, several studies have successfully separated different GSLs by adjusting sample preparation techniques, including variations in extraction solvents, concentrations, and durations [8]. Additionally, myrosinase treatment procedures are often incorporated into the extraction process for biological activity studies to enhance the levels of active products, such as sulforaphane [11].

Broccoli (Brassica oleracea var. italica) is notable for its anti-cancer properties, attributed to GSLs, which also influence flavor [12] and disease [13] and pest [14] resistance. Glucoraphanin (GRA), an aliphatic GSL, and its degradation product, sulforaphane (SF), are potent, safe, plant-derived anti-cancer compounds [15]. SF has shown promise in cancer prevention and treatment, influencing cell cycle regulation [16], acting as an anti-inflammatory component [17], and reducing oxidative stress [18]. Dietary SF from plant sources is considered non-toxic, is well-tolerated by the body, and is a naturally occurring substance suitable for clinical studies [19]. Existing research has shown significant variations in GRA content among cruciferous vegetables, with broccoli being the richest source [20].

The broccoli industry in China is undergoing rapid development and boasts the largest broccoli planting area worldwide. Despite this growth, there is an urgent need to improve the independent innovation capacity of the domestic broccoli seed industry. Currently, germplasm resources of broccoli with high anticancer potential and rich GRA content are relatively scarce. Therefore, it is critical to comprehensively characterize the GSL content and diversity of broccoli germplasm and to establish a comprehensive GSL database. This will facilitate the breeding of GRA-rich cultivars. In this study, we investigated the chemical diversity, content, and spatial–temporal distribution of GSLs among 191 broccoli genotypes. The results enhance the understanding of the content and distribution of GSLs in broccoli, providing a valuable resource and scientific evidence for targeted molecular breeding of GRA-rich broccoli.

2. Materials and Methods

2.1. Planting and Collection of Plant Materials

Broccoli samples were cultivated and harvested at the Wenzhou Academy of Agricultural Sciences, located in Tengqiao town (longitude: 120.51° E, latitude: 28.06° N, altitude: 10 m). The planted fields featured cyanoge clay soil, a predominant soil type for triple-cropping paddy fields in southern Zhejiang Province [21]. A total of 191 genotypes of broccoli, obtained through mating crosses over the past decade, were selected for this study. For each genotype, one hundred seeds were planted, and 30 uniformly growing plants were chosen. Ten plants were combined into a single sample, lyophilized, and ground to create a representative specimen. The collected samples encompassed various plant organs, including roots (at the four-euphylla stage), leaves (at the four-euphylla stage), stalks (at the four-euphylla stage), rosette leaves (at the rosette stage), rosette leaves (at the squaring stage), rosette leaves (at the floret stage), and florets (see Figure S1).

2.2. Sample Preparation

GSLs were extracted with minor modifications to the existing protocol [22]. Approximately 30 mg of freeze-dried sample was crushed using a JXFSTPRP-CLN freezing grinder (Jingxin, Shanghai, China) and then mixed with 1.5 mL of 70% methanol and 25 μL of 1.25 mmol/L Glucotropaeolin (Sigma, Dallas, TX, USA), and finally heated at 80 °C for 10 min, followed by centrifugation at 9000 rpm to collect the supernatant. We repeated the extraction process with hot methanol two times. The combined liquid was purified using a DEAE Sephadex A-25 anion-exchange column, which was washed with 2 mL 70% methanol (twice), 5 mL UPH₂O (twice), and 2 mL 20 mmol/L acetic acid buffer (pH = 5). Desulfurization was performed overnight with 0.5 mL of sulfatase (Sigma, St. Louis, MO, USA). The desulfurized samples were washed with 500 μL UPH₂O three times, followed by vacuum-drying using Concentrator plus (Eppendorf, Hamburg, Germany). The sample was then dissolved in 100 μL UPH₂O for analysis.

2.3. Qualitative and Quantitative Analyses of GSLs via UPLC-DAD

The current standard method for glucosinolate analysis involves desulfurizing glucosinolates with sulfatase and then employing an RP-HPLC gradient system for analysis [23]. The content and composition of GSLs were determined via UPLC analysis of the desulfoglucosinolates (dGSLs) following a previously described method [24]. The UPLC analysis was performed using an Agilent 1290 UPLC system equipped with a Waters ACQUITY UPLC HSS T3 column (50 mm × 2.1 mm, 1.8 µm), coupled with photodiode array detection. A gradient elution program was employed with solvent A (water) and solvent B (methanol) as the mobile phase, according to Table S1. The sample holder temperature was maintained at 4 °C during the analysis, while the column temperature was set at 30 °C. The flow rate and injection volume were set at 0.5 mL/min and 5 µL, respectively. The UV detection wavelength was set at 229 nm according to previous studies [25]. Quantification of dGSLs was based on integrative peak areas normalized against a known amount of an internal standard, glucotropaeolin.

The dGSLs, previously analyzed via UPLC as described above, underwent LC–MS analysis for qualitative assessment. LC–MS was performed using an Exion LC system coupled with a quadrupole time-of-flight Triple TOF 5600 mass spectrometer (Sciex, Foster City, CA, USA). The LC conditions and chromatographic column remained consistent with the quantitative analysis method. The mass spectrometer operated in positive ESI mode with optimized MS parameters set as follows: ion spray voltage at 5500 V; turbo spray temperature at 550 °C; curtain gas at 35 psi; nebulizer gas at 55 psi; heater gas at 55 psi; declustering potential at 80 V; collision energy at 25 eV; collision energy spread at 10 eV.

2.4. Statistical Analysis

All assays were conducted in triplicate. The data were processed using SPSS (ver. 22.0). One-way ANOVA and Tukey’s multiple-range test were used to evaluate significant differences (p < 0.05). Correlation analysis, K-mean analysis, and cluster analysis were performed using the OmicStudio tools at https://www.omicstudio.cn/tool (accessed on 20 August 2023) to assess the difference and relationships among GSLs across genotypes and organs in broccoli.

3. Results

3.1. Identifications of GSLs Based on Triple-TOF

Zhejiang Province, a major production and export hub for broccoli in China during winter and spring, is in need of a new form of germplasm development with high GRA content to enhance market competitiveness. In this study, we characterized the florets of 191 broccoli genotypes, identifying 13 glucosinolate compounds in mature florets (Table 1). Meanwhile, modifications to the extraction process of dGSLs (refer to Section 2) and their detection using the UPLC system enhanced separation efficiency and reduced detection time. GSL annotation was performed based on a comprehensive corpus of analytical information from previously reported GSLs and our previously published method [1,24]. This included an accurate mass determination with m/z values (<1 ppm) that aligned with theoretical m/z from published GSL data, as well as reasonable fragmentation patterns and diagnostic neutral loss of 162 Da, corresponding to an anhydroglucose of GSLs [1]. In total, we identified 13 GSLs, comprising seven aliphatic GSLs (glucoiberin (GIB), progoitrin (PRO), sinigrin (SIN), glucoraphanin (GRA), glucoalyssin (GAL), glucoibervirin (GIV), and glucoerucin (GER)); five indole GSLs (glucobrassicin (GBS), 4-methoxyglucobrassicin (4MGBS), neoglucobrassicin (NGBS), 4-hydroxyglucobrassicin (4HGBS), and 1-hydroxyglucobrassicin (1HGBS)); and one aromatic GSL (gluconasturtiin (GNT)) (Figure 1). Table 1 summarizes the retention time, measured masses, calculated masses, accurate masses, mass errors, and MS2 diagnostic ions for the identified dGSLs. Additionally, the MS2 spectrum and mass fragmentation patterns of dGSLs are presented in Figures S2–S4.

3.2. Evaluation of GSLs in Florets Based on 191 Broccoli Genotypes

After establishing the chemical diversity of GSLs in all the samples, we proceeded to quantitatively analyze the content of each component. The results reveal that aliphatic GSLs comprised 45.1%, indole GSLs 53.2%, and aromatic GSLs 1.7% of total GSLs (Figure 2A). Except for SIN, GIV, 1HGBS, GER, and GNT, the remaining eight GSLs were detected in all samples (Table S2), with GBS and GRA being the most abundant (Figure 2B). The GRA content ranged from 0.0260 to 3.1545 mg/g (DW), and GBS from 0.0144 to 1.5767 mg/g (DW). The total GSL content in the 191 broccoli samples ranged from 0.1705 to 5.8174 mg/g (DW), with the highest GSL content being approximately 34 times greater than the lowest (Figure 2C), aligning with previous studies [26]. The Q300 material displayed the highest total GSL and GRA content. We also analyzed the correlation between GSL content and agronomic traits like floret diameter and growth period (Table S3), finding no significant correlations (0.07 between floret diameter and GSL content; 0.17 between growth period and GSL content).

3.3. Evaluation of Total GSLs in Different Organs and Developmental Stages in Broccoli

The content of GSLs in plants is influenced by various factors, including genotype, tissue location, and developmental stage. Therefore, we assessed GSL content across genotypes, organs, and growth stages. K-means analysis clustered the 191 materials based on their GSL content, with the elbow method suggesting k = 3 for optimal clustering (Figure 3A). The materials were categorized into high (14 samples), middle (69 samples), and low (108 samples) GSL content groups (Figure 3B). For a more detailed investigation, we randomly selected six representative samples from each group to examine the GSL content across various organs and developmental stages. The findings revealed higher GSL content in roots, stalks, and leaves during the seedling stage (four-euphylla stage), especially in roots (Figure 3C). However, the GSL content in rosette leaves was relatively low during the rosette, squaring, and floret stages. Notably, florets in the “High” group matched or surpassed root GSL content at the four-euphylla stage. Additionally, the content of GSLs in leaves decreased initially and then increased from the seedling stage to the rosette stage, squaring stage, and floret stage. Overall, florets in the “High” group exhibited relatively high GSL content, presenting a valuable natural GSL source.

3.4. Analysis of GSL Diversity in Different Materials

The initial step in breeding broccoli varieties with high GSL content involves evaluating the distribution and content of GSLs in available broccoli materials. Among all GSLs, glucoraphanin (GRA) has received considerable attention due to its notable anticancer properties. We conducted a detailed analysis of the diversity and content of GSLs in various materials. All samples contained GSLs such as GAL, PRO, GBS, and 4MGBS. However, the remaining nine GSLs were not detected in some organs or developmental stages (see Figure 4). For instance, the rosette leaves during the rosette stage in both the “High” and “Low” groups, as well as those in the squaring stage in the “Middle” group, did not contain glucoiberin (GIB). During the seedling stage, roots exhibited higher levels of GER, 4MGBS, and NGBS, while stalks contained higher levels of GRA, and leaves had higher levels of both GRA and GBS. Overall, there was significant diversity and variation in the distribution of GSLs among roots, stalks, and leaves during the seedling stage (four-euphylla stage), as well as among rosette leaves at different developmental stages (rosette, squaring, and floret stages). This indicates that both the types and content of GSLs vary throughout the entire lifecycle of broccoli.

4. Discussion

In this study, 13 GSLs were identified among 191 broccoli genotypes, aligning with previous research that identified a similar range of GSLs in various numbers of genotypes. The 13 GSLs covered 10 of 12 GSLs identified in 80 broccoli genotypes [26], and 11 of 12 GSLs identified in 6 broccoli genotypes [27]. Notably, our findings predominantly feature aliphatic and indolyl GSLs, with a minor proportion of aromatic GSLs. In contrast, studies exploring GSL diversity in Brassica napus [28], Brassica oleracea var. capitata [29], Brassica pekinensis Rupr. [30], and other Brassica species [31,32,33] revealed a relatively higher proportion of aromatic GSLs, indicating a distinct species preference in GSL distribution. The low proportion of aromatic GSLs in broccoli (about 1%) and the inverse correlation observed between the content of aliphatic and indole GSLs, especially between GRA (aliphatic GSLs) and GBS (indole GSLs), are significant (see Figure 2C). The in vivo synthesis pathways of GSLs have been extensively elucidated, with the core structure being synthesized first [34,35], followed by side chain modification [36,37,38,39]. Research has demonstrated that the synthesis of GSLs is governed by a core complex comprising MYB and bHLH transcription factors (TFs). Notably, MYB TFs are divided into two distinct clades, each specifically regulating the synthesis of either aliphatic or indole GSLs [40]. Future studies could potentially enhance the yield of GRA (aliphatic GSLs) in broccoli via upregulating MYB28/29/76 expression and downregulating MYB34/51/122 expression, steering the core structure toward the aliphatic side chain modification pathway.

The total GSL content in broccoli florets from this study ranged from 0.1705 to 5.8174 mg/g (Figure 2), which is not exceptionally high compared to other cruciferous plants, such as canola (2.9 ± 0.9 mg/g), rapeseed (6.4 ± 1.3 mg/g), and turnip (14 ± 3.4 mg/g) [33]. However, the GRA content in broccoli (0.03 to 3.15 mg/g) is significantly higher than in other cruciferous plants like Chinese cabbage, which contains approximately 0 to 0.46 mg/g [30]. This observation aligns with findings from other studies examining broccoli leaves, which report GRA levels ranging from 0.11 to 0.79 mg/g [27]. In a separate study, Wang et al. treated the florets of ‘Yanxiu’ and ‘Xianglv No. 3’ with methyl Jasmonate, resulting in a GRA content of 1.7 mg/g, lower than that found in our GRA-rich genotypes [41]. Additionally, our research indicates high GSL content in broccoli seedlings, especially in roots, which is consistent with previous findings [26]. However, the limited biomass of seedlings makes the cost of consuming or extracting natural GSLs high. In contrast, the florets of materials with high GSL content offer a more practical option.

5. Conclusions

Conclusively, this study contributes significantly to the GSL database, providing one of the largest datasets to date. The identification of broccoli genotypes with high GSL content, particularly in florets, offers valuable resources for future research and breeding efforts. These high-GSL-content varieties could serve as excellent parental lines for breeding broccoli rich in GSLs. Additionally, comparing genotypes and gene expression between high- and low-GSL-level varieties could further our understanding of GSL genetics and the regulation of GSL-related genes. This research not only adds to our knowledge of GSL distribution in broccoli but also opens avenues for future genetic and breeding work to enhance GSL content, particularly GRA, for its health-promoting properties.

Author Contributions

M.Y.: Validation; Investigation; Data curation; Writing—original draft; C.S.: Validation; Investigation; Data curation; Writing—original draft; S.S.: Formal analysis; Investigation; Resources; Data curation; Writing—original draft; J.L.: Formal data analysis; Investigation; Data curation; Validation; Z.H.: Methodology; Validation; Investigation; S.L.: Conceptualization; Writing—review and editing; H.Z.: Conceptualization; Project administration; Z.T.: Investigation; Resources; Writing—review and editing; X.Y.: Conceptualization; Funding acquisition; Resources. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

We thank Haihong Wen (National and Local Joint Engineering Research Center) for her assistance with the experiments.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

GSLs	Glucosinolates
SF	Sulforaphane
UPH2O	Ultrapure Water
RP-HPLC	Reversed-Phase High-Performance Liquid Chromatography
HRMS	High-Resolution Mass Spectrum
dGSLs	Desulfoglucosinolates
LC–MS	Liquid Chromatograph–Mass Spectrometer
UPLC	Ultra Performance Liquid Chromatography
UPLC–UV–MS	Ultra Performance Liquid Chromatography–Ultraviolet–Mass Spectrometry
ESI	Electrospray Ionization
SPSS	Statistical Product and Service Solutions
ANOVA	Analysis of Variance
GIB	Glucoiberin
PRO	Progoitrin
SIN	Sinigrin
GRA	Glucoraphanin
GAL	Glucoalyssin
GIV	Glucoibervirin
GER	Glucoerucin
GBS	Glucobrassicin
4MGBS	4-Methoxyglucobrassicin
NGBS	Neoglucobrassicin
4HGBS	4-Hydroxyglucobrassicin
1HGBS	1-Hydroxyglucobrassicin
GNT	Gluconasturtiin
SEE	Sum of Squared Errors
DW	Dry Weight

Footnotes

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Figures and Table

Figure 1. Structures of desulfoglucosinolates found in Broccoli. Aliphatic dGSLs: glucoiberin (GIB), progoitrin (PRO), sinigrin (SIN), glucoraphanin (GRA), glucoalyssin (GAL), glucoibervirinn (GIV), and glucoerucin (GER). Indole dGSLs: glucobrassicin (GBS), 4-methoxyglucobrassicin (4MGBS), neoglucobrassicin (NGBS), 4-hydroxyglucobrassicin (4HGBS), and 1-hydroxyglucobrassicin (1HGBS). Aromatic dGSLs: glucotropaeolin (GTP), gluconasturtiin (GNT).

Figure 2. Concentration and diversity of GSLs in broccoli florets across 191 genotypes. (A) The average percentage of 13 GSLs distributed in all 191 materials. (B) Diverse distribution of GSLs in the 191 broccoli samples. (C) Statistical content of the total GSLs, GRA, and GBS in 191 broccoli samples.

Figure 3. Concentration of GSLs in different organs and developmental stages in broccoli. (A) Relationship between K-value and SEE (sum of the squared errors). (B) K-means analysis of total glucosinolates in 191 materials. All materials were divided into “High” (blue), “middle” (red), and “low” (green) groups according to their GSL contents. The detailed k-mean cluster information is listed in Table S3. (C) The contents of GSLs in different organs and at different developmental stages of 18 broccoli genotypes, including roots (R), stalks (S), leaves (L), rosette-stage rosette leaves (Y), squaring-stage rosette leaves (X), floret-stage rosette leaves (H), and florets (Q). The X-axis represents the number of the broccoli.

Figure 4. Diversity and content analyses of GSLs in broccoli from three clusters (High, Middle, and Low). The X-axis represents the species of GSLs. Q represents florets, H represents floret-stage rosette leaves, Y represents rosette-stage rosette leaves, X represents squaring-stage rosette leaves, L represents leaves, S represents stalks, and R represents roots.

Table 1

Identification of GSLs in broccoli.

Retention Time (min)	Typical Name	Type of Side Chain	Subcategory	Formula (Desulfo)	Diagnostic Ions (m/z)	Precursor Mass	Found at Mass	Mass Error (ppm)
1.13	Glucoiberin	(Rs)-3-(Methylsulfinyl)propyl	Aliphatic	C11H21NO7S2	182.0299, 118.0316	344.083	344.0833	0.3
1.41	Progoitrin	(2R)-2-Hydroxybut-3-enyl	Aliphatic	C11H19NO7S	148.0416, 130.0310, 85.0138	310.095	310.0958	0.9
1.68	Sinigrin	Prop-2-enyl	Aliphatic	C10H17NO6S	145.0482, 118.0310, 85.0269	280.085	280.0849	−0.2
1.81	Glucoraphanin	(Rs)-4-(Methylsulfiny)lbutyl	Aliphatic	C12H23NO7S2	196.0445, 132.0461, 87.0252	358.099	358.0990	0.4
3.00	Glucoalyssin	(Rs)-5-(Methylsulfinyl)pentyl	Aliphatic	C13H25NO7S2	210.0622, 142.0688	372.115	372.1148	0.8
3.53	4-Hydroxyglucobrassicin	4-Hydroxyindol-3-ylmethyl	Indole	C16H20N2O7S	223.0531, 190.0325, 146.0592	385.106	385.1066	0.4
4.14	Glucoibervirin	3-(Methylthio)propyl	Aliphatic	C11H21NO6S2	166.0340, 118.0303, 85.0273	328.088	328.0885	0.7
5.24	1-Hydroxyglucobrassicin	1-Hydroxyindolyl-3-methyl	Indole	C16H20N2O7S	223.0539, 190.0325, 146.0601	385.106	385.1064	0.1
5.28	Glucotropaeolin	Benzyl	Aromatic	C14H19NO6S	168.0469, 134.0595, 91.0527	330.101	330.1007	0.3
5.64	Glucoerucin	4-(Methylthio)butyl	Aliphatic	C12H23NO6S2	180.0495, 162.0398, 132.0461	342.104	342.1039	−0.3
6.05	Glucobrassicin	Indol-3-ylmethyl	Indole	C16H20N2O6S	207.0568, 174.0360, 130.0631	369.111	369.1112	−0.8
7.28	Gluconasturtiin	Phenethyl	Aromatic	C15H21NO6S	182.0632, 130.0639, 105.0688	344.116	344.1166	0.9
7.77	4-Methoxyglucobrassicin	4-Methoxyindol-3-ylmethyl	Indole	C17H22N2O7S	237.0691, 160.0747	399.122	399.1220	−0.1
8.54	Neoglucobrassicin	N-Methoxyindol-3-ylmethyl	Indole	C17H22N2O7S	237.0677, 206.0487, 130.0625	399.122	399.1223	0.7

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13122928/s1, Figure S1: The collected samples of broccoli from 191 genotypes; Figure S2: MS2 spectrum of aliphatic desulfoglucosinolates in broccoli; Figure S3: MS2 spectrum of indole desulfoglucosinolates in broccoli; Figure S4: MS2 spectrum of aromatic desulfoglucosinolates in broccoli; Table S1: The gradient program for UPLC; Table S2: Qualitative and quantitative analyses of GSLs in the florets of 191 broccoli materials; Table S3: Diameter and growth stage information of broccoli florets; Table S4: The contents of GSLs in different organs and at different developmental stages of 18 broccoli genotypes. Table S5: Weather information for Tengqiao town.

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