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Article

Glucosinolate Content in Brassica Genetic Resources and Their Distribution Pattern within and between Inner, Middle, and Outer Leaves

1
National Agrobiodiversity Center, National Institute of Agricultural Sciences, RDA, Jeonju 54874, Korea
2
Client Service Division, Planning and Coordination Bureau, RDA, Jeonju 54875, Korea
3
National Creative Research Laboratory for Ca2+ signaling Network, Jeonbuk National University Medical School, Jeonju, 54896, Korea
4
Jeonbuk Agricultural Research and Extension Services, Iksan 54591, Korea
*
Author to whom correspondence should be addressed.
Plants 2020, 9(11), 1421; https://doi.org/10.3390/plants9111421
Submission received: 18 September 2020 / Revised: 15 October 2020 / Accepted: 22 October 2020 / Published: 23 October 2020

Abstract

:
Glucosinolates (GSLs) are sulfur-containing secondary metabolites naturally occurring in Brassica species. The purpose of this study was to identify the GSLs, determine their content, and study their accumulation patterns within and between leaves of kimchi cabbage (Brassica rapa L.) cultivars. GSLs were analyzed using UPLC-MS/MS in negative electron-spray ionization (ESI) and multiple reaction monitoring (MRM) mode. The total GSL content determined in this study ranged from 621.15 to 42434.21 μmolkg−1 DW. Aliphatic GSLs predominated, representing from 4.44% to 96.20% of the total GSL content among the entire samples. Glucobrassicanapin (GBN) contributed the greatest proportion while other GSLs such as glucoerucin (ERU) and glucotropaeolin (TRO) were found in relatively low concentrations. Principal component analysis (PCA) yielded three principal components (PCs) with eigenvalues ≥ 1, altogether representing 74.83% of the total variation across the entire dataset. Three kimchi cabbage (S/No. 20, 4, and 2), one leaf mustard (S/No. 26), and one turnip (S/No. 8) genetic resources were well distinguished from other samples. The GSL content varied significantly among the different positions (outer, middle, and inner) of the leaves and sections (top, middle, bottom, green/red, and white) within the leaves. In most of the samples, higher GSL content was observed in the proximal half and white sections and the middle layers of the leaves. GSLs are regarded as allelochemicals; hence, the data related to the patterns of GSLs within the leaf and between leaves at a different position could be useful to understand the defense mechanism of Brassica plants. The observed variability could be useful for breeders to develop Brassica cultivars with high GSL content or specific profiles of GSLs.

1. Introduction

Glucosinolates (GSLs), also called β-thioglucoside-N-hydroxysulfates, are a class of sulfur-containing important plant secondary metabolites naturally occurring in Brassica species [1]. GSLs are most frequently classified as aliphatic, aromatic, and indole GSLs based on the structure of their side chain (R group). The side chain is mainly derived from amino acid precursors including methionine (and also alanine, leucine, isoleucine, or valine in some cases) for aliphatic, phenylalanine for aromatic, and tryptophan for indole GSLs [2]. However, this classification wrongly used the names aliphatic, aromatic, and indole as synonyms of methionine, phenylalanine-, and tryptophan-derived GSLs, respectively [3]. Blažević et al. [3] presented a more meaningful classification alternative to that previously used: (i) based on the amino acid precursor (tryptophan-derived versus isoleucine-derived versus methionine-derived); (ii) according to the type of degradation product (stable isothiocyanate-yielding versus thiocyanate ion-yielding versus oxazolidine-2-thione yielding GSLs); and (iii) according to the presence or absence of an aromatic moiety in the GSL. Most GSLs share a basic chemical structure consisting of a β-D-glucopyranose residue linked via a sulfur atom to a (Z)-N-hydroximinosulfate ester and a variable R group [4]. Glucosinolates and their degradation products exhibit wide ranges of biological activities, including both negative and positive nutritional attributes and the mediation of plant–herbivore interactions. Upon hydrolysis by myrosinases, GSLs produce several degradation products, such as isothiocyanates, thiocyanates, oxazolidinthiones, epithionitriles, and nitriles [5]. GSLs and their biosynthetic products are implicated to reduce the risk of cancer in humans [6,7,8] and exhibit antimicrobial activities [9,10,11]. The health-related functions of GSLs are dictated by their bioavailability. GSLs and their degradation products undergo transformation, assimilation, absorption, and elimination after ingestion in the human gut [12,13]. Although their contribution is complex to understand, GSLs are also regarded as an important component of flavor in cooked vegetables [14]. GSLs and their degradation products mediate the process of plant defense mechanism against danger by serving as a feeding deterrent to a wide range of herbivores such as birds, mammals, mollusks, aquatic invertebrates, nematodes, bacteria, and fungi [14,15,16]. Contrary, the same GSLs attract and stimulate specialist herbivores such as the larvae of the lepidopteran species Plutella xylostella and Pieris rapae [15], which often use these compounds as cues for feeding or oviposition. The biocidal activity of GSL-containing Brassica plants has made them a promising alternative to synthetic pesticides for pest and disease control [17,18]. In planta studies of various Brassica seedlings have also shown a positive correlation between GSL content and disease severity [19].
GSLs are reported to be found in the vegetative and reproductive tissues of various dicotyledonous plant families and are the major secondary metabolites in mustard-oil plants of the Brassicaceae family [4,20]. The main food sources of glucosinolates are reviewed by Possenti et al. [21]. The content of GSLs accounts for around 1% of the dry weight in Brassica vegetables and can reach up to 10% in the seeds of some plants [4]. The qualitative and quantitative profiles of total and individual GSLs in Brassica vegetables vary significantly due to several factors such as cultivar genotype [22,23], developmental stage [24], environmental conditions (temperature, light, water, and soil) [25,26,27,28], growing seasons [29], agricultural practices [30], level of insect damage [27,31], and post-harvest conditions [32]. Wide geographic and evolutionary variation is recorded in broccoli [29], A. thaliana [33], Chinese cabbage [23], and cabbage (B. oleracea L.) [34]. Apart from the aforementioned factors, GSLs tend to vary quantitatively and qualitatively based on plant part, as observed in kale [27], in cabbage [26], and A. thaliana [24].
Commonly, GSLs are extracted using boiling water/methanol followed by desulfonation of intact GSLs on Sephadex-A25 columns [34], followed by quantitation and identification by HPLC. However, the desulfonation process has been found to be laborious and time-consuming [35], and some GSLs could be insufficiently desulfonated in the process at a lower concentration of sulfatase [36]. GC-MS methods are often used for detailed analysis [37]. Recently, a simplified method of sample extraction from lyophilized samples followed by quantitation and identification of intact GSLs using UPLC-DAD-MS/MS in multiple reaction monitoring (MRM) mode was reported [38].
Leaves of kimchi cabbage, turnip, mibuna, leaf mustard, and cabbage are commonly used for various dishes in many countries. Kimchi cabbage is a major ingredient in kimchi and a widely consumed traditional fermented food in Korea [23]. Several comparative studies on the profiles of GSLs in Brassica germplasm collections across the world are available in the literature [1,22,29,39,40,41]. However, most of the studies so far are focused on the levels of GSLs in the seeds of Brassica plants [9,22,42]. Lee et al. [23] identified and quantified ten different GSLs in breed varieties of kimchi cabbage collected from the Republic of Korea. Studies on diverse collections of genetic resources such as gene bank germplasm collections are elusive. Reports about the variability of GSLs on leaves of B. rapa L. were also given less attention compared to seeds. Yang and Quiros [43] found extensively varied GSL content among B. rapa L varieties. Accessions from a Russian gene data bank showed a wide variety of GSLs qualitatively and quantitatively among the genetic resources [44]. Another study on varieties of turnip greens from Spain also showed wide diversity in the quality and quantity of GSLs [45]. The wide range of variability in the type and amount of GSLs from different countries [44,45,46], in addition to other experimental related factors, could underline the variability in the GSL biosynthesis pathway within the plant to adapt the surrounding conditions. Many plant natural products, including GSLs, serve as defenses against herbivores [31]. It is important to determine the GSL content in different tissues of the plant to understand the actual defense role that a potential herbivore would encounter. In this study, we have identified and quantified eight GSLs, namely gluconapin, glucobrassicanapin, progoitrin, glucotropaeolin, glucoerucin, gluconasturtiin, glucoberteroin, and glucobrassicin, in 48 genetic resources including kimchi cabbage (B. rapa L.), turnip (B. rapa L.), mibuna (B. rapa L.), leaf mustard (Brassica juncea L. Czern.), and cabbage (B. oleracea L.) collected from China, Ethiopia, Japan, North Korea, South Korea, and Taiwan. The crops were grown in uniform agricultural conditions. Moreover, the spatial accumulation patterns of GSLs within and between the leaves of three kimchi cabbage commercial cultivars have been determined.

2. Materials and Methods

2.1. Reagents and Standards

All chemicals and solvents used during extraction and analysis were of analytical grade and purchased from Fisher Scientific Korea Ltd. (Seoul, South Korea) and Sigma-Aldrich (St. Louis, MO, USA). GSL standards (gluconapin, glucobrassicanapin, progoitrin, glucotropaeolin, glucoerucin, gluconasturtiin, glucoberteroin, and glucobrassicin) were purchased from Phytoplan Diehm & Neuberger GmbH (Heidelberg, Germany). All individual GSL standards had purity greater than or equal to 97%.

2.2. Plant Materials

The seeds of 48 genetic resources (43 germplasm collections and five commercial cultivars), belonging to B. rapa L., Brassica juncea L. Czern., and B. oleracea L. and originating from six different countries (China (13), Ethiopia (1), Japan (1), North Korea (1), South Korea (12), and Taiwan (20)) were obtained from the gene bank of South Korea and grown at the research farm of the National Agrobiodiversity Center (NAC), Jeonju (35°49′18″ N 127°08′56″ E), Republic of Korea. Seeds were sown in plug trays in the last week of August, 2018, and seedlings were grown inside a greenhouse. After a month, healthy-looking seedlings (4 to 5 leaves) were transplanted to an area of 60 × 40 cm per plant in an experimental field of NAC. Harvesting was conducted in the first week of November. Plant cultural practices were followed as per the recommendation of the Rural Development Administration (RDA) of South Korea. Fertilizers (N-K-P-Ca-B = 65-45-100-100-1.5 kg/10a) were applied before transplanting the seedlings followed by RDA’s standard, and drip irrigation tape was used for watering. One teaspoon per plant of nitrogen fertilizer was applied when the plant started to form bulbs (12–14 leaves). Each accession consisted of 25 plants. Plant growth was maintained using nutrient solution throughout the growing season. As external damage could alter the content of GSLs, the plant materials were protected from any damage, and 10 to 15 healthy plants were used for sampling for the analysis of GSLs. Leaves were collected from the outer, inner, and middle location of each plant and mixed. In each accession, three replicate samples were prepared. Great care was taken to prevent thawing of the sample to minimize enzymatic degradation of GSLs. Samples were immediately frozen and all equipment in contact with them was held at subzero temperatures until further processing.
To study the GSL spatial distribution within sections of the leaf of kimchi cabbage and between leaves, two green-pigmented (“Hangamssam” and “Alchandul”) and one red-pigmented (“Bbalgang3-ho”) commercial cultivars were selected. The inner, middle, and outer leaves were separated. Each leaf was then dissected into the top, middle, bottom, green/red, and white parts as required. Three replicates were prepared from 15 healthy plants accordingly. Sampling positions of kimchi cabbage plants are shown in Figure 1. Additional information about the germplasm collections and commercial cultivars is presented in Table 1.

2.3. Sample Pretreatment, Extraction, and Analysis of GSLs

Samples were harvested, placed in a vinyl freezer bag, and kept at −80 °C until further processing. The frozen samples were subsequently lyophilized for 48 h using LP500 vacuum freeze-drier (Ilshinbiobase Co., Seoul, Korea), ground to fine powder, and kept at −80 °C until analysis. The extraction of GSLs was conducted following the method reported by Ishida et al. (2011) [47]. Briefly, 0.1 g sample was mixed with 5 mL of 80% methanol, held at 25 °C for 30 min, and shaken at 120 r/min for 30 min at room temperature. The mixture was centrifuged using VS-180CFi centrifuge (Vision Scientific Co., Daejeon, Korea) (centrifuge conditions set at 14,000 rpm, 4 °C, and 10 min). The supernatant was transferred into a vial and GSLs were analyzed immediately using UPLC-MS/MS.
Intact GSLs were analyzed using an Acquity UPLC System (Waters, Milford, MA, USA) coupled to Xevo™ TQ-S system (Waters, MS Technologies, Manchester, UK). Chromatographic separation was carried out using Acquity UPLC BEH C18 (1.7 μm, 2.1 × 100 mm) column (Waters Corp., Manchester, UK). The flow rate was kept at 0.5 mL/min; the column temperature was maintained at 35°C, and the injection volume was 5 μL. The mobile phase was composed of 0.1% trifluoroacetic acid in water as eluent A and 0.1% trifluoroacetic acid in methanol as eluent B. The elution conditions were as follows: initial condition set at 100% of A; 0.0–1.0 min, 100% of A; 1.0–7.0 min, 100 to 80% A; 7.0–10 min, 80 to 0% of A; 10–11 min, 0 to 100% of A; 11–15 min, 100% of A. The mass spectrometry instrument was operated in negative ion electrospray ionization (ESI) and multiple reaction monitoring (MRM) mode. Data acquisition was performed using MassLynx 4.1 software. For MS/MS detection, the ionization source parameters were set as follows: the capillary and con voltages were set as 3kV and 54 v, respectively; the ion source and the desolvation temperatures were set as 150 and 350 °C, respectively. The cone and desolvation gas were set at flow rates of 150 and 650 Lh−1, respectively. GSLs were identified by comparing their retention times and MS and MS/MS fragmentation spectra with those of commercial standards. Individual GSLs were quantified by MRM, considering one MS/MS transition for each compound. Selected transitions and other MRM parameters are presented in Table 2. The final concentration of individual GSLs was calculated using linear regression equations derived from the calibration curves of the corresponding standards. Results were calculated from peak area responses and presented as µmolkg−1 sample dry weight (DW).
The established UPLC-MS/MS method of analysis was validated by measuring the linear, intraday, and interday precision. Standard stock solutions of glucosinolates were prepared by dissolving 10 mg in methanol to obtain a final concentration of 1 mg/mL. Standard calibration curves that were used to quantify the GSLs were prepared from serially diluted solutions (1000 to 1 ng/mL) from the stock solution. Calibration curve parameters are presented in Table 2. The precision of the method was determined as the percentage of the ratio of the standard deviation to the mean value (relative standard deviation, RSD) of interday and intraday analysis. Both precision and accuracy of the method were within the acceptable limit of ± 15% of the actual values. The limit of detection (LOD) and limit of quantification (LOQ) values were determined as, respectively, three and ten times the standard error of the intercept of the regression equation of the linear calibration curve divided by the slope. Based on the residual standard deviation of the response and the slope, the LODs for the nine GSLs ranged between 0.5 and 1 ng/mL, and LOQs were between 1.5 and 3 ng/mL. Test solutions were prepared freshly before analysis.

2.4. Statistical Analysis

Results were expressed as mean ± standard deviation (SD) of triplicates. The data were treated with analysis of variance (ANOVA) followed by Duncan’s multiple range test (p < 0.05) using the SPSS V. 17.0 statistical program (SPSS Inc., Chicago, USA). Principal component analysis (PCA) was performed using the statistical program R (Rstudio, Inc., Austria). Data were visualized using principal components score and loading plots (PCA-Biplot). Points represented an individual sample, and the lines represented the contribution of an individual GSL to the score.

3. Results and Discussion

In this study, eight GSLs were identified and quantified in leaves of five commercial varieties and 45 germplasm collections of Brassica plants belonging to B. rapa L., B. juncea L. Czern., and B. oleracea L. The concentrations of GSLs were also evaluated in various leaf sections and positions of two green- (“Hangamssam” and “Alchandul”) and a red- (“Bbalgang 3-ho”) pigmented commercial varieties commonly called kimchi cabbage. Five aliphatic (GNA, GBN, PRO, ERU, and BER), two phenylalkyl (TRO and NAS), and one indole (GBC) GSLs were identified. GSLs were examined using negative ionization electrospray (ESI) LC-MS/MS in MRM mode by monitoring specific transitions originating the characteristic fragment ions (Table 2). The results of this study, presented and discussed in detail in the next sections, showed that the values varied widely among the entire germplasm collections and between different sections and positions of the Brassica leaves. Principal component analysis (PCA) was employed to identify the GSL exhibiting the greatest variance across the entire collection and to determine closely related individual GSLs.

3.1. Variation in GSL Content between Germplasm Collections

As can be seen in Table 3, a significant difference in GSL content was observed among the germplasm collections and commercial varieties of Brassica plants. The total GSL content ranged from 621.15 (“Alchandul”, S/No. 42) to 42,434.21 (IT 260822, S/No. 2) µmol kg−1 DW with an average value of 14,050.97 µmol kg−1 DW. Aliphatic GSLs were dominant throughout the entire collections, which altogether represented from 4.44% to 96.2% (average 66.12%) of the total GSL content, followed by phenylalkyl GSLs (0.90%~81.32%; average 17.56%). GBC, the only indole GSL detected in our study, represented as low as 1.36% and as high as 69.59% of the total GSLs. GBN (0.04~23,026.64 µmol kg−1 DW), representing an average of 45.06% was the most dominant GSL across the entire collections. GNA (11.90 ~ 15,276.50 µmol kg−1 DW), GBC (120.81~12,134.40 µmol kg−1 DW), and NAS (46.60 ~ 6353.11 µmol kg−1 DW), representing an average 13.47%, 16.31%, and 17.37%, respectively, represented a moderate proportion. The least dominant GSLs were BER, PRO, ERU, and TRO and presented average values of 433.35, 426.15, 52.17, and 13.46 µmol kg−1 DW in the entire samples, respectively. Some accessions were found to accumulate unusually high content of a particular type of GSL. For example, one turnip (S/No 8) and four kimchi cabbage genetic resources (S/No. 10, 13, 22, and 27) contained more than 90% aliphatic glucosinolates. The highest amount of GBC, the only indole GSL detected, was detected in one cabbage (S/No. 45) and two kimchi cabbage germplasm (S/No. 20 and 35). Accession 47 (IT 100409) had the highest content of phenylalkyl GSL (81.32%), where NAS being contributed most. Accessions 12 (IT 228167) and 20 (IT 32750) had the highest ERU and GBC content, respectively, accounting about 3.5-fold higher than the accessions containing the second-highest in the entire sample.
Most of the accessions were originated from Taiwan (20), China (13), and South Korea (12). Taiwanese originated Brassica resources exhibited higher averaged combined GSL content (16, 392 µmol kg−1 DW), followed by Chinese (15, 794 µmol kg−1 DW) and South Korean (8,156 µmol kg−1 DW) originated resources. In terms of individual glucosinolates, Taiwanese originated resources had the highest GNA, GBN, TRO, and ERU, while Chinese originated materials excel in PRO, NAS, and GBC levels. South Korean originated resources were superior in their BER content. The PCA plot of the first two components showed that the genotypes were distributed throughout the four quadrants with no significant grouping based on their country of origin, suggesting the absence of intrinsic similarities between them in their GSL content based on their origin (Supplementary File, Figure S2).
GNA and GBN were documented as the most abundant GSLs in the leaves of B. napa, as reported previously [23,44,45,48]. However, GBN, 4-methoxyglucobrassicin, and PRO were dominant in the same crop in another study [49]. The identity and quantity of GSLs vary considerably between various crops of Brassica. For example, the predominant GSLs in broccoli were glucoraphanin, GNA, and GBC, while sinigrin was found to be the dominant GSL in green cabbage, Brussels sprouts, cabbage, cauliflower, and kale [22,34]. This study revealed a wide variety of GSLs among accessions of Brassica germplasm collections. The difference observed in the GSL profile is both qualitative and quantitative. This could determine their level of nutritional and health-promoting properties and supports the feasibility of developing cultivars with an enhanced level of GSLs through genetic manipulation. Previous studies showed the impact of temperature [27], amount of rainfall [50], radiation [51,52], plant part examined [1], phenological stage of growth [24,27], and level of insect damage [27,53] on the level of GSLs.
Other Brassica plant leaf sources of the GSLs investigated in our study include but are not limited to broccoli, Brussels sprout, cauliflower, kale, Chinese cabbage, rocket plants, pak choi, and watercress [48,54,55,56,57,58,59,60,61,62,63,64]. We compared the levels of GSLs in other Brassica plants in previous reports that employed LC/MS and LC/MS/MS methods. The most dominant GSL in this study, GBN, was previously reported in the ranges of 970 to 10,480 µmol/kg DW [46] and 400 to 8080 µmol/kg DW [48] in Chinese cabbage and from 2.52 to 20.19 µmol/kg DW in pak choi [55]. The GNA levels ranged from 250 to 11,100 µmol/kg DW [46] and 400 to 8990 µmol/kg DW [48] in Chinese cabbage, which were in agreement with our study. However, compared to our results, quite higher (4910 to 70,670 µmol/kg DW) and lower (ND to 340 µmol/kg DW) levels were recorded in pak choi [55] and rocket [63], respectively. Comparable levels of PRO were obtained in rocket (187.4 to 624.7 µmol/kg DW) [62] and Chinese cabbage (140 to 3520 µmol/kg DW) [46] to our study. Pak choi contained high PRO (1160 to 41,510 µmol/kg DW) compared to other Brassica plants in previous reports and this study [55]. Broccoli (379.2 to 2895.2 µmol/kg DW), Brussels sprouts (14,92.9 to 2532.6 µmol/kg DW), cauliflower (655.5 to 2887.6 µmol/kg DW) [59], kale (3200 to 7250 µmol/kg DW) [57], Chinese cabbage (130 to 6810 µmol/kg DW) [48], and pak choi (880 to 4860 µmol/kg DW) [55] contained moderately comparable levels of GBC to our samples, while rocket (17.8 to 44.6 µmol/kg DW) had a significantly lower amount [62]. Watercress had high levels of NAS (4155.8 µmol/kg DW) [56] compared to other Brassica plants but in corcondance with this study. ERU was recorded as a dominant GSL in rocket [58,60,61,62,63,64] and much higher compared to this study, but comparable results were obtained in pak choi (ND to 2370 µmol/kg DW) [55] and Chinese cabbage (40 to 750 µmol/kg DW) [48].

3.2. Intra- and Inter-Leaf Distribution of GSLs in Kimchi Cabbage

The leaves of three green-/red-pigmented kimchi cabbage cultivars including “Hangamssam” (green), “Alchandul” (green), and “Bbalgang 3-ho” (red) were segregated based on their position in the whole plant as inner, middle, and outer layers. Each leaf was further portioned into different sections (top, middle, bottom, green/red, and white). The GSL content in kimchi cabbage significantly varied based on leaf section, position, and color. The GSL content in different leaf sections/positions of the three kimchi cabbage cultivars is presented in Figure 2 and Supplementary File (Table S1). The leaf parts were sampled as demonstrated in Figure 1. The white section within the leaf contained a higher total sum of GSLs (1.52 to 33.07-fold higher) than the green/red section, except in the outer layer of “Bbalgang 3-ho”, where the red section contained a 3.31-fold higher total GSL concentration than the white section. The trend in total GSL content within different leaf sections (top, middle, and bottom) was not strictly consistent. However, in most cases, higher GSL content was observed at the proximal half of the leaves. Concerning the position of the leaf (outer, middle, and inner layers) in the whole plant, the average total GSL content in the middle layers was 1.34-, 1.42-, and 3.21-fold higher than in the outer layers of “Hangmassam”, “Alchandul”, and “Bbalgang 3-ho” cultivars, respectively. The content of total GSLs evaluated in the inner layers of “Alchandul” and “Bbalgang 3-ho” showed no significant difference with the outer layers. In general, the middle layer leaves were found to contain higher concentrations of GSLs compared to older (outer) leaves and the younger inner layer leaves. The green-pigmented cultivars showed superiority in total glucosinolate content over the red-pigmented cultivars. In an earlier study, the inner layers of B. oleracea var. capitate leaves were reported to exhibit 1.1- to 1.8-fold higher GSL concentrations than the outer positions [65]. In another study, younger leaves of Raphanus sativus were found to contain higher GSL content [66].
The enhancement of GSL concentration upon plant damage [53] has long indicated that GSLs are plant defense chemicals where mostly their defensive properties are attributed to the toxicity and deterrence nature of their degradation products [15]. In contrast, there are also cases where GSLs mediated by their volatile hydrolysis products could serve to attract adapted herbivores that often use GSLs as cues for feeding or oviposition [15]. The spatial distribution of GSLs in different sections of a single leaf and/or location of the leaf in the whole plant could partly be important to explain the patterns of herbivory. Studies devoted to GSL spatial patterns within leaves of kimchi cabbage are elusive. The proximal halves of R. sativus leaves contained a higher mean concentration of GSLs compared to the distal halves of leaves [66]. Shroff et al. (2008) [67] studied the spatial distribution (midvein, inner lamina, and outer lamina) of GSLs in leaves of A. thaliana and tried to relate the distribution to the pattern of herbivory caused by larvae of the lepidopteran, Helicoverpa armigera. These authors found out that the GSL abundance in the inner vs. the peripheral part of the leaf affected insect feeding preference and anti-herbivore defenses. As stated in the previous section and shown in Figure 2, the white part (midvein) of kimchi cabbage contained relatively higher GSLs compared to the green- or red-colored part. This is consistent with A. thaliana leaves, where the midvein part exhibited the greatest concentration compared to the other sections of the leaf [67]. This could be due to the distribution of certain biosynthetic enzymes exclusively to vascular bundles [68], resulting in greater synthesis and storage of GSLs in the midvein (white part) of the leaf of kimchi cabbage. It could also be related to ecological significance as the midvein is critical to the function of the leaf, and the transport of water and nutrients takes place through it [69]. The greater concentration of GSL in the white part of kimchi cabbage in our study corroborates the idea of the storage of GSLs being associated with the vascular system. The higher content of GSLs in the middle (younger) leaves compared to the outer (older) leaves in this study is also in agreement with the predictions of optimal defense theory: younger leaves are more valuable as they have higher future photosynthetic potential and need a higher degree of protection from damage [70]. In addition, GSL concentration could tend to decrease in outer leaves due to the dilution of GSLs as the leaf expands [70].

3.3. Multivariate Analysis

The results of PCA are indicated by the principal components score and loading plots (PCA-Biplot). The PCA of GSL data yielded three principal components with eigenvalues ≥1, accounting for 74.83% of the total variance across the entire dataset. The first, second, and third principal components (PCs) contributed 37.47%, 20.88%, and 16.47% of the total variance, respectively. The loadings, eigenvalue, and percentage of variance obtained for all principal components (PCs) are presented in the Supplementary File (Table S2). Scores and loading plots of the first two PCs obtained from GSL content of 48 Brassica germplasm collections are presented in Figure 3. The loadings of GSLs (represented by light blue arrows) show the extent and nature of each GSL concentration contribution to the principal components. All the GSLs were positively correlated with PC1, while GNA, GBN, and NAS had a positive correlation with PC2. NAS was the predominant GSL in PC1, followed by PRO, GBN, and BER, while GNA, GBN, GBC, and BER had a major contribution to PC2, with the last two affecting it negatively. Three kimchi cabbage (S/No. 20, 4, and 2, the former located at the bottom right and the latter two at the top right quadrant of the PCA plot), one leaf mustard (S/No. 26, located at the top right quadrant of the PCA plot), and one turnip (S/No. 8, located at the top left quadrant of the PCA plot) genetic resources were seen well distinguished from other samples. The separation of S/No. 20 and S/No. 4 from other accessions in the score plot could be described by their significantly higher content of GBC and NAS, respectively. On the other hand, S/No. 2 (IT260822) had relatively high content of NAS and GBN (ranked second and third) compared to other genetic resources. S/No. 26 is characterized by its high content of GBN and GNA (ranked first and third in the entire collections, respectively) while S/No. 8 had the highest concentration of GNA in the entire collection of genetic resources.

4. Conclusions

Eight GSLs were identified and quantified in Brassica germplasm collections and commercial varieties using the UPLC-MS/MS method in multiple reaction monitoring scan mode. Remarkable differences in total and individual GSLs were observed among different samples. The data in this study revealed a wide variation in the level of GSLs among genotypes, leaf position/section, and leaf color. The PCA in this study allowed easy visualization of the data, and five genetic resources (S/No. 20, 4, 2, 26, and 8) were seen separated from the entire collections. The inter- and intra-leaf variations of GSLs were examined in three commercial kimchi cabbage varieties. The GSL content varied significantly among leaves in different positions of the plant (outer, middle, and inner) and sections within leaves (top, middle, bottom, green/red, and white). Higher GLS content was observed in the proximal half and white sections of the leaves and middle layers in all of the samples tested. The variation in the GSL level suggests that the potential health benefits of Brassica plants could depend on the type of accession used. The wide variability observed in GSL content among the germplasm collections in this study offers important and basic information for enhancing the level of GSLs in Brassica plants through breeding and hence their health beneficial properties. Besides this, the development of Brassica plants with specific GSL profiles of specific health beneficial properties would help for a meaningful recommendation of dietary intake of Brassica vegetables. Two aliphatic (GBN and GNA), one phenylalkyl (NAS), and one indole (GBC) were detected in relatively higher amount compared to other GSLs. As the breakdown products of these GSLs are implicated to posses antimicrobial, antibacterial, and anticancer properties elsewhere, they could be used as potential biomarkers for the consumption of kimchi cabbage. In this study, we determined the variability of GSL content and composition reflected between Brassica genetic resources and within and between leaves. The results would widen the present understanding of the accumulation pattern of GSLs in leaves of Brassica plants and provide information about the nature of plant defenses towards a perceived danger.

Supplementary Materials

The following are available online at https://www.mdpi.com/2223-7747/9/11/1421/s1, Figure S1: A representative MRM profile of the mixture of glucosinolates. Figure S2: Principal component analysis (PCA) plot of the scores generated based on the country of origin for 45 samples and nine glucosinolates (Chinese origin, 13 accessions; South Korean origin, 12 accessions; and Taiwanese origin, 20 accessions). Table S1: Glucosinolate concentration in different leaf sections of three cultivars of kimchi cabbage (µmol kg−1 DW). Table S2: Loadings, eigenvalues, and percentage of variance for the principal components (PCs) data from germplasm collections.

Author Contributions

Conceptualization, J.-H.R., H.-C.K., J.-J.N. and A.D.A.; Data curation, S.C., O.-S.H. and A.-J.H.; Formal analysis, J.-H.R., S.C., Y.-J.C. and A.D.A.; Funding acquisition, J.-H.R.; Methodology, Y.-J.C.; Resources, H.-C.K.; Validation, J.-E.L., N.-Y.R., A.-J.H. and Y.-J.C.; Writing—original draft, A.D.A.; Writing—review and editing, J.-H.R., J.-E.L., O.-S.H., N.-Y.R., J.-J.N. and A.D.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was carried out with the support of the “Research Program for Agricultural Science & Technology Development (Project NO. PJ01425501)”, National Institute of Agricultural Sciences, Rural Development Administration, Republic of Korea.

Conflicts of Interest

All authors have no conflict of interest to declare. The funder had no role in the design of the study, in the collection of, analysis, or interpretation of the data, in writing the manuscript, or in the decision to publish the results.

References

  1. Bhandari, S.R.; Jo, J.S.; Lee, J.G. Comparison of glucosinolate profiles in different tissues of nine Brassica crops. Molecules 2015, 20, 15827–15841. [Google Scholar] [CrossRef]
  2. Ishida, M.; Hara, M.; Fukino, N.; Kakizaki, T.; Morimitsu, Y. Glucosinolate metabolism, functionality and breeding for the improvement of Brassicaceae vegetables. Breed. Sci. 2014, 64, 48–59. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Blažević, I.; Montaut, S.; Burčul, F.; Olsen, C.E.; Burow, M.; Rollin, P.; Agerbirk, N. Glucosinolate structural diversity, identification, chemical synthesis and metabolism in plants. Phytochemistry 2019, 169, 112100. [Google Scholar] [CrossRef] [PubMed]
  4. Fahey, J.; Zalcmann, A.; Talalay, P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 2001, 56, 5–51. [Google Scholar] [CrossRef]
  5. Vig, A.P.; Rampal, G.; Thind, T.S.; Arora, S. Bio-protective effects of glucosinolates-A review. LWT Food Sci. Technol. 2009, 42, 1561–1572. [Google Scholar] [CrossRef]
  6. Hong, E.; Kim, G.-H. Anticancer and antimicrobial activities of β-phenylethyl isothiocyanate in Brassica rapa L. Food Sci. Technol. Res. 2008, 14, 377–382. [Google Scholar] [CrossRef] [Green Version]
  7. Kim, H.; Kim, J.-Y.; Kim, H.-J.; Kim, D.-K.; Jo, H.-J. Anticancer activity and quantitative analysis of glucosinolates from green and red leaf mustard. Korean J. Food Nutr. 2011, 24, 362–366. [Google Scholar] [CrossRef]
  8. Catanzaro, E.; Fimognari, C. Antileukemic activity of sulforaphane. In Glucosinolates; Mérillon, J.M., Ramawat, K.G., Eds.; Springer: Cham, Switzerland, 2017; pp. 301–317. [Google Scholar]
  9. Blažević, I.; Radonić, A.; Skočibušić, M.; Denicola, G.R.; Montaut, S.; Iori, R.; Rollin, P.; Mastelić, J.; Zekić, M.; Maravić, A. Glucosinolate profiling and antimicrobial screening of Aurinia leucadea (Brassicaceae). Chem. Biodivers. 2011, 8, 2310–2321. [Google Scholar] [CrossRef]
  10. Tierens, K.; Thomma, B.; Brouwer, M.; Schmidt, J.; Kistner, K.; Porzel, A.; Mauch-Mani, B.; Cammue, B.; Broekaert, W. Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens. Plant Physiol. 2001, 125, 1688–1699. [Google Scholar] [CrossRef] [Green Version]
  11. Saladino, F.; Bordin, K.; Luciano, F.B.; Franzón, M.F.; Mañes, J.; Meca, G. Antimicrobial activity of the glucosinolates. In Glucosinolates; Mérillon, J.M., Ramawat, K.G., Eds.; Springer: Cham, Switzerland, 2017; pp. 249–274. [Google Scholar]
  12. Barba, F.J.; Nikmaram, N.; Roohinejad, S.; Khelfa, A.; Zhu, Z.; Koubaa, M. Bioavailability of glucosinolates and their breakdown products: Impact of processing. Front. Nutr. 2016, 3, 24. [Google Scholar] [CrossRef] [Green Version]
  13. Sørensen, J.C.; Frandsen, H.B.; Jensen, S.K.; Kristensen, N.B.; Sørensen, S.; Sørensen, H. Bioavailability and in vivo metabolism of intact glucosinolates. J. Funct. Foods 2016, 24, 450–460. [Google Scholar] [CrossRef]
  14. Mithen, R. Glucosinolates—Biochemistry, genetics and biological activity. Plant Growth Regul. 2001, 34, 91–103. [Google Scholar] [CrossRef]
  15. Halkier, B.A.; Gershenzon, J. Biology and biochemistry of glucosinolates. Annu. Rev. Plant Biol. 2006, 57, 303–333. [Google Scholar] [CrossRef] [Green Version]
  16. Singh, A. Glucosinolates in plant defense. In Glucosinolates; Mérillon, J.M., Ramawat, K.G., Eds.; Springer: Cham, Switzerland, 2017; pp. 237–246. [Google Scholar]
  17. Lazzeri, L.; Baruzzi, G.; Malaguti, L.; Antoniacci, L. Replacing methyl bromide in annual strawberry production with glucosinolate-containing green manure crops. Pest Manag. Sci. 2003, 59, 983–990. [Google Scholar] [CrossRef]
  18. Bangarwa, S.K.; Norsworthy, J.K. Glucosinolate and isothiocyanate production for weed control in plasticulture production system. In Glucosinolates; Mérillon, J.M., Ramawat, K.G., Eds.; Springer: Cham, Switzerland, 2017; pp. 201–235. [Google Scholar]
  19. Aires, A.; Dias, C.S.; Carvalho, R.; Oliveira, M.H.; Monteiro, A.A.; Simões, M.V.; Rosa, E.A.; Bennett, R.N.; Saavedra, M.J. Correlations between disease severity, glucosinolate profiles and total phenolics and Xanthomonas campestris pv. campestris inoculation of different Brassicaceae. Sci. Hortic. 2011, 129, 503–510. [Google Scholar] [CrossRef]
  20. Rodman, J.E.; Soltis, P.S.; Soltis, D.E.; Sytsma, K.J.; Karol, K.G. Parallel evolution of glucosinolate biosynthesis inferred from congruent nuclear and plastid gene phylogenies. Am. J. Bot. 1998, 85, 997–1006. [Google Scholar] [CrossRef] [Green Version]
  21. Possenti, M.; Baima, S.; Raffo, A.; Durazzo, A.; Giusti, A.M.; Natella, F. Glucosinolates in food. In Glucosinolates; Mérillon, J.M., Ramawat, K.G., Eds.; Springer: Cham, Switzerland, 2017; pp. 87–132. [Google Scholar]
  22. Kushad, M.M.; Brown, A.F.; Kurilich, A.C.; Juvik, J.A.; Klein, B.P.; Wallig, M.A.; Jeffery, E.H. Variation of glucosinolates in vegetable crops of Brassica oleracea. J. Agric. Food Chem. 1999, 47, 1541–1548. [Google Scholar] [CrossRef]
  23. Lee, M.K.; Chun, J.H.; Byeon, D.H.; Chung, S.O.; Park, S.U.; Park, S.; Arasu, M.V.; Al-Dhabi, N.A.; Lim, Y.P.; Kim, S.J. Variation of glucosinolates in 62 varieties of Chinese cabbage (Brassica rapa L. ssp. pekinensis) and their antioxidant activity. LWT Food Sci. Technol. 2014, 58, 93–101. [Google Scholar] [CrossRef]
  24. Brown, P.D.; Tokuhisa, J.G.; Reichelt, M.; Gershenzon, J. Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana. Phytochemistry 2003, 62, 471–481. [Google Scholar] [CrossRef]
  25. Khan, M.; Ulrichs, C.; Mewis, I. Influence of water stress on the glucosinolate profile of Brassica oleracea var. italica and the performance of Brevicoryne brassicae and Myzus persicae. Entomol. Exp. Appl. 2010, 137, 229–236. [Google Scholar] [CrossRef]
  26. Rosa, E.A.S.; Rodrigues, P.M.F. The effect of light and temperature on glucosinolate concentration in the leaves and roots of cabbage seedlings. J. Sci. Food Agric. 1998, 78, 208–212. [Google Scholar] [CrossRef]
  27. Velasco, P.; Cartea, M.E.; González, C.; Vilar, M.; Ordás, A. Factors affecting the glucosinolate content of kale (Brassica oleracea acephala group). J. Agric. Food Chem. 2007, 55, 955–962. [Google Scholar] [CrossRef]
  28. Volden, J.; Borge, G.I.A.; Bengtsson, G.B.; Hansen, M.; Thygesen, I.E.; Wicklund, T. Effect of thermal treatment on glucosinolates and antioxidant-related parameters in red cabbage (Brassica oleracea L. ssp. capitata f. rubra). Food Chem. 2008, 109, 595–605. [Google Scholar] [CrossRef]
  29. Brown, A.F.; Yousef, G.G.; Jeffery, E.H.; Klein, B.P.; Wallig, M.A.; Kushad, M.M.; Juvik, J.A. Glucosinolate profiles in broccoli: Variation in levels and implications in breeding for cancer chemoprotection. J. Amer. Soc. Hort. Sci. 2002, 127, 807–813. [Google Scholar] [CrossRef]
  30. Rosen, C.J.; Fritz, V.A.; Gardner, G.M.; Hecht, S.S.; Carmella, S.G.; Kenney, P.M. Cabbage yield and glucosinolate concentrations as affected by nitrogen and sulfur fertility. HortScience 2005, 40, 1493–1498. [Google Scholar] [CrossRef] [Green Version]
  31. Ulmer, B.J.; Dosdall, L.M. Glucosinolate profile and oviposition behavior in relation to the susceptibilities of Brassicaceae to the cabbage seedpod weevil. Entomol. Exp. Appl. 2006, 121, 203–213. [Google Scholar] [CrossRef]
  32. Park, M.-H.; Arasu, M.V.; Park, N.-Y.; Choi, Y.-J.; Lee, S.-W.; Al-Dhabi, N.A.; Kim, J.B.; Kim, S.-J. Variation of glucoraphanin and glucobrassicin: Anticancer components in Brassica during processing. Food Sci. Technol. Camp. 2013, 33, 624–631. [Google Scholar] [CrossRef] [Green Version]
  33. Windsor, A.J.; Reichelt, M.; Figuth, A.; Svatoš, A.; Kroymann, J.; Kliebenstein, D.J.; Gershenzon, J.; Mitchell-Olds, T. Geographic and evolutionary diversification of glucosinolates among near relatives of Arabidopsis thaliana (Brassicaceae). Phytochemistry 2005, 66, 1321–1333. [Google Scholar] [CrossRef]
  34. Park, S.; Valan Arasu, M.; Lee, M.K.; Chun, J.H.; Seo, J.M.; Lee, S.W.; Al-Dhabi, N.A.; Kim, S.J. Quantification of glucosinolates, anthocyanins, free amino acids, and vitamin C in inbred lines of cabbage (Brassica oleracea L.). Food Chem. 2014, 145, 77–85. [Google Scholar] [CrossRef]
  35. Tian, Q.; Rosselot, R.A.; Schwartz, S.J. Quantitative determination of intact glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Anal. Biochem. 2005, 343, 93–99. [Google Scholar] [CrossRef]
  36. Hennig, K.; Verkerk, R.; Bonnema, G.; Dekker, M. Pitfalls in the desulphation of glucosinolates in a high-throughput assay. Food Chem. 2012, 134, 2355–2361. [Google Scholar] [CrossRef]
  37. Matthäus, B.; Fiebig, H. Simultaneous determination of isothiocyanates, indoles, and oxazolidinethiones in myrosinase digests of rapeseeds and rapeseed meal by HPLC. J. Agric. Food Chem. 1996, 44, 3894–3899. [Google Scholar] [CrossRef]
  38. Gratacós-Cubarsí, M.; Ribas-Agustí, A.; García-Regueiro, J.A.; Castellari, M. Simultaneous evaluation of intact glucosinolates and phenolic compounds by UPLC-DAD-MS/MS in Brassica oleracea L. var. botrytis. Food Chem. 2010, 121, 257–263. [Google Scholar] [CrossRef]
  39. Bodnaryk, R.P.; Palaniswamy, P. Glucosinolate levels in cotyledons of mustard, Brassica juncea L. and rape, B. Napus L. do not determine feeding rates of flea beetle, Phyllotreta cruciferae (goeze). J. Chem. Ecol. 1990, 16, 2735–2746. [Google Scholar] [CrossRef]
  40. Griffiths, D.W.; Deighton, N.; Birch, A.E.; Patrian, B.; Baur, R.; Städler, E. Identification of glucosinolates on the leaf surface of plants from the Cruciferae and other closely related species. Phytochemistry 2001, 57, 693–700. [Google Scholar] [CrossRef]
  41. Velasco, L.; Becker, H.C. Variability for seed glucosinolates in a germplasm collection of the genus Brassica. Genet. Resour. Crop Evol. 2000, 47, 231–238. [Google Scholar] [CrossRef]
  42. Song, L.; Morrison, J.J.; Botting, N.P.; Thornalley, P.J. Analysis of glucosinolates, isothiocyanates, and amine degradation products in vegetable extracts and blood plasma by LC-MS/MS. Anal. Biochem. 2005, 347, 234–243. [Google Scholar] [CrossRef]
  43. Yang, B.; Quiros, C.F. Survey of glucosinolate variation in leaves of Brassica rapa crops. Genet. Resour. Crop Evol. 2010, 57, 1079–1089. [Google Scholar] [CrossRef] [Green Version]
  44. Klopsch, R.; Witzel, K.; Artemyeva, A.; Ruppel, S.; Hanschen, F.S. Genotypic variation of glucosinolates and their breakdown products in leaves of Brassica rapa. J. Agric. Food Chem. 2018, 66, 5481–5490. [Google Scholar] [CrossRef]
  45. Padilla, G.; Cartea, M.E.; Velasco, P.; de Haro, A.; Ordas, A. Variation of glucosinolates in vegetable crops of Brassica rapa. Phytochemistry 2007, 68, 536–545. [Google Scholar] [CrossRef]
  46. Baek, S.A.; Jung, Y.H.; Lim, S.H.; Park, S.U.; Kim, J.K. Metabolic profiling in Chinese cabbage (Brassica rapa L. subsp. pekinensis) cultivars reveals that glucosinolate content is correlated with carotenoid content. J. Agric. Food Chem. 2016, 64, 4426–4434. [Google Scholar] [CrossRef]
  47. Ishida, M.; Kakizaki, T.; Ohara, T.; Morimitsu, Y. Development of a simple and rapid extraction method of glucosinolates from radish roots. Breed. Sci. 2011, 61, 208–211. [Google Scholar] [CrossRef] [Green Version]
  48. Kim, J.K.; Chu, S.M.; Kim, S.J.; Lee, D.J.; Lee, S.Y.; Lim, S.H.; Ha, S.H.; Kweon, S.J.; Cho, H.S. Variation of glucosinolates in vegetable crops of Brassica rapa L. ssp. pekinensis. Food Chem. 2010, 119, 423–428. [Google Scholar] [CrossRef]
  49. Hong, E.; Kim, S.J.; Kim, G.H. Identification and quantitative determination of glucosinolates in seeds and edible parts of Korean Chinese cabbage. Food Chem. 2011, 128, 1115–1120. [Google Scholar] [CrossRef]
  50. Ciska, E.; Martyniak-Przybyszewska, B.; Kozlowska, H. Content of glucosinolates in cruciferous vegetables grown at the same site for two year under different climatic conditions. J. Agric. Food Chem. 2000, 48, 2862–2867. [Google Scholar] [CrossRef] [PubMed]
  51. Banerjee, A.; Variyar, P.S.; Chatterjee, S.; Sharma, A. Effect of post harvest radiation processing and storage on the volatile oil composition and glucosinolate profile of cabbage. Food Chem. 2014, 151, 22–30. [Google Scholar] [CrossRef] [PubMed]
  52. Rybarczyk-Plonska, A.; Hagen, S.F.; Borge, G.I.A.; Bengtsson, G.B.; Hansen, M.K.; Wold, A.B. Glucosinolates in broccoli (Brassica oleracea L. var. italica) as affected by postharvest temperature and radiation treatments. Postharvest Biol. Technol. 2016, 116, 16–25. [Google Scholar] [CrossRef]
  53. Birch, A.N.E.; Wynne Griffiths, D.; Hopkins, R.J.; Macfarlane Smith, W.H.; McKinlay, R.G. Glucosinolate responses of swede, kale, forage and oilseed rape to root damage by turnip root fly (Delia floralis) larvae. J. Sci. Food Agric. 1992, 60, 1–9. [Google Scholar] [CrossRef]
  54. Smith, T.K.; Lund, E.K.; Clarke, R.G.; Bennett, R.N.; Johnson, I.T. Effects of Brussels sprout juice on the cell cycle and adhesion of human colorectal carcinoma cells (HT29) in vitro. J. Agric. Food Chem. 2005, 53, 3895–3901. [Google Scholar] [CrossRef]
  55. Wiesner, M.; Zrenner, R.; Krumbein, A.; Glatt, H.; Schreiner, M. Genotypic variation of the glucosinolate profile in pak choi (Brassica rapa ssp. chinensis). J. Agric. Food Chem. 2013, 61, 1943–1953. [Google Scholar] [CrossRef]
  56. Giallourou, N.; Oruna-Concha, M.J.; Harbourne, N. Effects of domestic processing methods on the phytochemical content of watercress (Nasturtium officinale). Food Chem. 2016, 212, 411–419. [Google Scholar] [CrossRef]
  57. Steindal, A.L.H.; Rdven, R.; Hansen, E.; Mlmann, J. Effects of photoperiod, growth temperature and cold acclimatisation on glucosinolates, sugars and fatty acids in kale. Food Chem. 2015, 174, 44–51. [Google Scholar] [CrossRef]
  58. Bell, L.; Oruna-Concha, M.J.; Wagstaff, C. Identification and quantification of glucosinolate and flavonol compounds in rocket salad (Eruca sativa, Eruca vesicaria and Diplotaxis tenuifolia) by LC-MS: Highlighting the potential for improving nutritional value of rocket crops. Food Chem. 2015, 172, 852–861. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Pellegrini, N.; Chiavaro, E.; Gardana, C.; Mazzeo, T.; Contino, D.; Gallo, M.; Riso, P.; Fogliano, V.; Porrini, M. Effect of different cooking methods on color, phytochemical concentration, and antioxidant capacity of raw and frozen Brassica vegetables. J. Agric. Food Chem. 2010, 58, 4310–4321. [Google Scholar] [CrossRef] [PubMed]
  60. Jin, J.; Koroleva, O.A.; Gibson, T.; Swanston, J.; Maganj, J.; Zhang, Y.A.N.; Rowland, I.R.; Wagstaff, C. Analysis of phytochemical composition and chemoprotective capacity of rocket (Eruca sativa and Diplotaxis tenuifolia) leafy salad following cultivation in different environments. J. Agric. Food Chem. 2009, 57, 5227–5234. [Google Scholar] [CrossRef] [PubMed]
  61. Kim, S.J.; Ishii, G. Glucosinolate profiles in the seeds, leaves and roots of rocket salad (Eruca sativa Mill.) and anti-oxidative activities of intact plant powder and purified 4-methoxyglucobrassicin. Soil Sci. Plant Nutr. 2006, 52, 394–400. [Google Scholar] [CrossRef]
  62. Pasini, F.; Verardo, V.; Caboni, M.F.; D’Antuono, L.F. Determination of glucosinolates and phenolic compounds in rocket salad by HPLC-DAD-MS: Evaluation of Eruca sativa Mill. and Diplotaxis tenuifolia L. genetic resources. Food Chem. 2012, 133, 1025–1033. [Google Scholar] [CrossRef]
  63. Villatoro-Pulido, M.; Priego-Capote, F.; Álvarez-Sánchez, B.; Saha, S.; Philo, M.; Obregón-Cano, S.; De Haro-Bailón, A.; Font, R.; Del Río-Celestino, M. An approach to the phytochemical profiling of rocket [Eruca sativa (Mill.) Thell]. J. Sci. Food Agric. 2013, 93, 3809–3819. [Google Scholar] [CrossRef] [PubMed]
  64. Taranto, F.; Francese, G.; Di Dato, F.; D’Alessandro, A.; Greco, B.; Onofaro Sanajà, V.; Pentangelo, A.; Mennella, G.; Tripodi, P. Leaf metabolic, genetic, and morphophysiological profiles of cultivated and wild rocket salad (Eruca and Diplotaxis Spp.). J. Agric. Food Chem. 2016, 64, 5824–5836. [Google Scholar] [CrossRef]
  65. Choi, S.H.; Park, S.; Lim, Y.P.; Kim, S.J.; Park, J.T.; An, G. Metabolite profiles of glucosinolates in cabbage varieties (Brassica oleracea var. capitata) by season, color, and tissue position. Hortic. Environ. Biotechnol. 2014, 55, 237–247. [Google Scholar] [CrossRef]
  66. Shelton, A.L. Within-plant variation in glucosinolate concentrations of Raphanus sativus across multiple scales. J. Chem. Ecol. 2005, 31, 1711–1732. [Google Scholar] [CrossRef]
  67. Shroff, R.; Vergara, F.; Muck, A.; Svatos, A.; Gershenzon, J. Nonuniform distribution of glucosinolates in Arabidopsis thaliana leaves has important consequences for plant defense. Proc. Natl. Acad. Sci. USA 2008, 105, 6196–6201. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  68. Reintanz, B.; Lehnen, M.; Reichelt, M.; Gershenzon, J.; Kowalczyk, M.; Sandberg, G.; Godde, M.; Uhl, R.; Palme, K. Bus, a bushy Arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain aliphatic glucosinolates. Plant Cell 2001, 13, 351–367. [Google Scholar] [CrossRef] [Green Version]
  69. Hamilton, J.; Zangerl, A.R.; DeLucia, E.H.; Berenbaum, M.R. The carbon-nutrient balance hypothesis: Its rise and fall. Ecol. Lett. 2001, 4, 86–95. [Google Scholar] [CrossRef] [Green Version]
  70. McCall, A.C.; Fordyce, J.A. Can optimal defence theory be used to predict the distribution of plant chemical defences? J. Ecol. 2010, 98, 985–992. [Google Scholar] [CrossRef]
Figure 1. Representative photos of sampling positions of kimchi cabbage based on (a) leaf sections: I, III, III refers to the upper, middle, and bottom parts of the leaf. The white section is indicated by the triangular dashed line. The green/red part was sampled from the whole leaf excluding the white section. (b) Location of the leaves in the whole plant: I, II, and III refer to the outer (two layers), middle (three layers), and inner (the remaining) parts of the vegetable.
Figure 1. Representative photos of sampling positions of kimchi cabbage based on (a) leaf sections: I, III, III refers to the upper, middle, and bottom parts of the leaf. The white section is indicated by the triangular dashed line. The green/red part was sampled from the whole leaf excluding the white section. (b) Location of the leaves in the whole plant: I, II, and III refer to the outer (two layers), middle (three layers), and inner (the remaining) parts of the vegetable.
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Figure 2. Glucosinolate levels in different leaf sections of three cultivars of kimchi cabbage: (a) Hangamssam; (b) Bbanlgang 3-ho; and (c) Alchandul.
Figure 2. Glucosinolate levels in different leaf sections of three cultivars of kimchi cabbage: (a) Hangamssam; (b) Bbanlgang 3-ho; and (c) Alchandul.
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Figure 3. Principal component analysis (PCA) plot of the scores (indicated by dotes) and loadings (indicated by lines) of the 48 Brassica plants based on the first and second principal components. The numbers 1–48 correspond to the S/No in Table 1 and Table 3. GNA = gluconapin; GBN = glucobrassicanapin; PRO = progoitrin; TRO = glucotropaeolin; ERU = glucoerucin; NAS = gluconasturtiin; BER = glucoberteroin; and GBC = glucobrassicin.
Figure 3. Principal component analysis (PCA) plot of the scores (indicated by dotes) and loadings (indicated by lines) of the 48 Brassica plants based on the first and second principal components. The numbers 1–48 correspond to the S/No in Table 1 and Table 3. GNA = gluconapin; GBN = glucobrassicanapin; PRO = progoitrin; TRO = glucotropaeolin; ERU = glucoerucin; NAS = gluconasturtiin; BER = glucoberteroin; and GBC = glucobrassicin.
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Table 1. Accession number, scientific name, common name, and origin of 48 germplasm accessions of Brassica genus.
Table 1. Accession number, scientific name, common name, and origin of 48 germplasm accessions of Brassica genus.
S/NoAccession No.Scientific Name *Crop NameGiven NameOriginClassification
1IT260816Brassica rapa L.Kimchi cabbageCHINA-YAAS-2010-103ChinaBreeding line
2IT 260822Brassica rapa L.Kimchi cabbageCHINA-YAAS-2010-109ChinaBreeding line
3IT 100390Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100390Taiwan-
4IT 260819Brassica rapa L.Kimchi cabbageCHINA-YAAS-2010-106ChinaBreeding line
5IT 100414Brassica rapa L.TurnipAVRDC-KJH-1985-100414Taiwan-
6IT 260820Brassica rapa L.Kimchi cabbageCHINA-YAAS-2010-107ChinaBreeding line
7IT 100416Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100416Taiwan-
8IT 100413Brassica rapa L.TurnipAVRDC-KJH-1985-100413Taiwan-
9IT 100388Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100388Taiwan-
10IT 100408Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100408Taiwan-
11IT 260824Brassica rapa L.Kimchi cabbageCHINA-YAAS-2010-111ChinaBreeding line
12IT 228167Brassica rapa L.Kimchi cabbage36197Taiwan-
13IT 100404Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100404Taiwan-
14IT 100352Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100352Taiwan-
15IT 293231Brassica rapa L.Kimchi cabbageWIR68EthiopiaCultivar
16IT 100412Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100412Taiwan-
17IT 100411Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100411Taiwan-
18IT 100371Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100371Taiwan-
19IT 135409Brassica rapa L.Kimchi cabbageShingatsunaJapanLandrace
20IT 32750Brassica rapa L.Kimchi cabbageChing Pao 26ChinaCultivar
21IT 100406Brassica rapa L.MibunaAVRDC-KJH-1985-100406Taiwan
22IT 100353Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100353Taiwan
23IT 100372Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100372Taiwan
24Commercial Brassica rapa L.Kimchi cabbageHangamssam 1South KoreaCultivar
25IT 100366Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100366Taiwan
26IT 100393Brassica rapa L.Leaf mustardAVRDC-KJH-1985-100393Taiwan
27IT 100395Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100395Taiwan
28IT 163625Brassica rapa L.Kimchi cabbageYeongdeog Sandongchae-2South KoreaLandrace
29Commercial Brassica rapa L.Kimchi cabbageWeoldongdaewangSouth KoreaCultivar
30IT 199678Brassica rapa L.Kimchi cabbageWIR33507ChinaLandrace
31IT 199706Brassica rapa L.Kimchi cabbageWIR30643ChinaLandrace
32IT 32733Brassica rapa L.Kimchi cabbageSong Dao Xin 2ChinaCultivar
33IT 32738Brassica rapa L.Kimchi cabbageWeonsi-1984-Kimchicabbage32738South Korea-
34IT 219574Brassica rapa L.Kimchi cabbageKang re jieqiuxiayangbaoxinbai 50 tianChinaCultivar
35IT 262102Brassica rapa L.Kimchi cabbageNamyeon1-hoNorth koreaCultivar
36IT 100383Brassica rapa L.Kimchi cabbageAVRDC-KJH-1985-100383Taiwan-
37IT 120112Brassica rapa L.Kimchi cabbageShuang Ching 156ChinaCultivar
38IT 163707Brassica rapa L.Kimchi cabbageJangsuSandongchaeSouth KoreaLandrace
39IT 166984Brassica rapa L.Kimchi cabbageTianjin qingChinaLandrace
40IT 163708Brassica rapa L.Kimchi cabbageMuju Sandongchae1South KoreaLandrace
41Commercial Brassica rapa L.Kimchi cabbageBalgang 3-hoSouth KoreaCultivar
42Commercial Brassica rapa L.Kimchi cabbageAlchandulSouth KoreaCultivar
43IT 215003Brassica rapa L.Kimchi cabbageJeonnam Haenam-2000-36South KoreaLandrace
44IT 199670Brassica rapa L.Kimchi cabbageDak-seSouth KoreaLandrace
45IT 206799Brassica oleracea L.CabbageNPL-KIG-1997-278South Korea-
46IT 216342Brassica rapa L.Kimchi cabbageBaoshou 3ChinaCultivar
47IT 100409Brassica juncea L. Czern.Leaf mustardAVRDC-KJH-1985-100409Taiwan-
48CommercialBrassica rapa L.Kimchi cabbageHangamssam2South KoreaCultivar
* Scientific names of each plant are assigned based on the status given on http://www.theplantlist.org. Only accepted names are used.
Table 2. List of identified glucosinolates, retention time (RT), calibration curves, and multiple reaction monitoring (MRM) conditions for quantitation of glucosinolates by negative ion MRM (see Supplementary File (Figure S1) for chromatogram).
Table 2. List of identified glucosinolates, retention time (RT), calibration curves, and multiple reaction monitoring (MRM) conditions for quantitation of glucosinolates by negative ion MRM (see Supplementary File (Figure S1) for chromatogram).
Glucosinolates RT (min)MRM Transition CID (ev)Dwell Time (sec) Calibration Curve Parameters
Progoitrin (PRO)1.41387.77 > 194.85 200.033Y = 3.59902X – 20.5808 (r2 = 0.999)
Gluconapin (GNA)3.02371.74 > 258.74 200.033Y = 3.50074X + 3.51886 (r2 = 0.996)
Glucobrassicanapin (GBN)4.42385.71 > 258.87 250.033Y = 2.68899X – 2.8434 (r2 = 0.994)
Glucotropaeolin (TRO)4.84407.72 > 258.87 200.033Y = 6.27084X – 4.49552 (r2 = 0.999)
Glucoerucin (ERU)4.97419.69 > 258.74 250.033Y = 2.41077X + 16.6315 (r2 = 0.999)
Glucobrassicin (GBC)5.61446.69 > 204.94 200.033Y = 1.76969X – 11.3033 (r2 = 0.999)
Glucoberteroin (BER)6.29433.72 > 275.06 200.033Y = 2.92616X – 3.54071 (r2 = 0.993)
Gluconasturtiin (NAS)6.33421.69 > 274.87 250.033Y = 1.98894X + 1.81048 (r2 = 0.994)
CID = collision-induced dissociation; LOQ = limit of quantification; Pol. = polarity.
Table 3. Glucosinolate content (µmol kg−1 DW) in 48 germplasm accessions of Brassica (n = 3).
Table 3. Glucosinolate content (µmol kg−1 DW) in 48 germplasm accessions of Brassica (n = 3).
S/NoGluconapinGlucobrassicanapin Progoitrin GlucotropaeolinGlucoerucin GluconasturtiinGlucoberteroinGlucobrassicinSum
159.88 ± 9.48A14,961.04 ± 64R1191.24 ± 46.86U21.08 ± 0.36P59.85 ± 4.55L3713.98 ± 118.91Q450.56 ± 8.05P1257.38 ± 46.19M21,715.00 ± 186.56Q
213,634.53 ± 32.45S19,279.35 ± 711.33V1548.95 ± 12.76X20.96 ± 1.63OP39.21 ± 2.26J6307.25 ± 365.08W664.07 ± 34.97R939.88 ± 77.28KL42,434.21 ± 1042.22X
324.07 ± 2.22A19,737.72 ± 527.7W1207.7 ± 23.17UV18.96 ± 1.38OP20.73 ± 0.51H4701.79 ± 91.65T247.37 ± 7.70K-M2352.8 ± 65.59Q28,311.15 ± 639.34V
454.11 ± 7.74A18,361.17 ± 307.39U1979.79 ± 43.03Y9.13 ± 0.46JK116.8 ± 2.76Q6353.11 ± 137.87W1221.33 ± 13.01T909.34 ± 15.88KL29,004.78 ± 212.28V
511.9 ± 0.61A5877.32 ± 85.72HI13.78 ± 0.19AB31.77 ± 1.12R10.3 ± 0.62E-G1070.32 ± 20.69HIJ17.19 ± 1.18AB375.17 ± 24.16C-E7407.74 ± 124.18FG
63476.23 ± 182.91lM13,029.46 ± 135.44P147.35 ± 5.72I6.21 ± 0.71F-I27.7 ± 2.97I5471.57 ± 147.53V310.33 ± 5.7NO633.73 ± 27.11G-I23,102.58 ± 347ST
74735.79 ± 147.57P17,678.49 ± 118.58T30.92 ± 2.36ABC8.14 ± 1.13IJ3.48 ± 0.31A-E1808.78 ± 49.56L52.03 ± 2.5A-F967.2 ± 9.61KL25,284.81 ± 204.38U
815,276.5 ± 3.34T6141.34 ± 63.04HI109.45 ± 6.7G8.91 ± 0.17JK3.55 ± 0.22A-E635.54 ± 15.93DEF16.21 ± 0.23AB413.84 ± 4.04D-F22,605.35 ± 44.95RS
93728.9 ± 164.19M13,101.21 ± 394.68P560.33 ± 11.65O15.61 ± 1.58N4.74 ± 0.23A-E3677.9 ± 184.07Q61.98 ± 1.17B-G1623.02 ± 116.35N22,773.70 ± 830.86R-T
102946.79 ± 232.69K9778.47 ± 286.18M32.95 ± 1.13A-D21.01 ± 0.89OP2.76 ± 0.2A-D918.89 ± 33.45GHI13.13 ± 1.09AB278.44 ± 8.22A-D13,992.44 ± 545.46L
113299.16 ± 59L9673.08 ± 198.58LM296.33 ± 10.91L2.3 ± 0.1A-C7.61 ± 0.17B-F4023.25 ± 75.16R160.08 ± 3.25I821.22 ± 19.58I-K18,283.02 ± 281.06O
123535.26 ± 199.95lM5062.22 ± 88.73GH12.64 ± 1.72AB7.87 ± 0.42H-J725.93 ± 10.93U3134.41 ± 121.18O2574.83 ± 88.13Y842.07 ± 20.2I-K15,895.23 ± 287.36M
134191.84 ± 46.19NO9971.01 ± 269.09M331.44 ± 19.07L14.61 ± 1.27MN3.25 ± 0.35A-E643.2 ± 12.04DEF78.21 ± 1.74C-H500.03 ± 11.73E-G15,733.6 ± 283.92M
143336.34 ± 165.05L13,541.59 ± 96.59Q474.05 ± 17.5N9.05 ± 1.17JK5.92 ± 0.2A-E2722.06 ± 114.26N85.32 ± 1.8D-H547.25 ± 7.77E-G20,721.58 ± 158.21P
153467.66 ± 112.06LM6626.38 ± 172.30J227.4 ± 4.68K4.03 ± 0.51B-F76.69 ± 0.99MN4261.8 ± 210.19S279.83 ± 7.15L-N603.83 ± 27.82F-H15,547.64 ± 447.31M
164010.97 ± 74.12N3497.18 ± 12.57EF165.63 ± 1.76I8.18 ± 0.28IJ85.21 ± 2.39O1773.78 ± 68.97L166.24 ± 2.24IJ1088.08 ± 34.99LM10,795.26 ± 175.08J
1711.93 ± 2.53A2924.5 ± 44.36DE111.5 ± 4.38GH29.87 ± 1.33R85.48 ± 6.99O858.03 ± 28.63F-H236.7 ± 11.87KL546.02 ± 33.92E-G4804.03 ± 115.95D
182070.69 ± 49J9270.47 ± 118.15L918.9 ± 12.68T20.1 ± 0.32OP125.37 ± 0.74R4700.27 ± 183.2T1616.48 ± 36.22V3206.66 ± 111.92S21,928.94 ± 399.73QR
1921.75 ± 4.52A15,704.45 ± 392.29S417.99 ± 1.94M11.73 ± 0.51L204.85 ± 6.86T4700.35 ± 145.32T457.36 ± 8.04P1953.32 ± 63.03O23,471.8 ± 375.26T
201190.43 ± 38.54FG6263.87 ± 114.31HIJ1474.34 ± 9.07W39.75 ± 0.45S106.73 ± 2.46P3441.64 ± 180.17P806.95 ± 12.97S12134.4 ± 474.88U25,458.11 ± 810.62U
212973.98 ± 57.69k10,636.42 ± 86.88N96.36 ± 4.52FG18.51 ± 0.65O3.26 ± 0.41A-E2264.32 ± 39.6M49.01 ± 2.14A-F647.84 ± 17.57G-J16,689.69 ± 163.39N
225144.32 ± 213.54q1101.59 ± 71.18B0.52 ± 0.05A12.04 ± 0.66L0.76 ± 0.06AB46.6 ± 5.15A0.19 ± 0.01A186.51 ± 12A-C6492.51 ± 297.72E
231602.12 ± 31.52hi10,590.99 ± 161.77N873.97 ± 13.86S29.87 ± 0.55R78.11 ± 5.31N5047.2 ± 100.45U1494.98 ± 35.08U2955.72 ± 34.85R22,672.94 ± 351.75R-T
242866.29 ± 596.67K9938.31 ± 179.31M626.3 ± 13.28P15.83 ± 0.68N188.01 ± 6.86S2814.85 ± 71.76N2426.77 ± 72.17W1256.82 ± 29.37M20,133.19 ± 869.75P
251266.69 ± 23.11FG11,840.19 ± 410.97O795.24 ± 21.51R57.1 ± 3.27U48.10 ± 1.20K5079.75 ± 283.32U663.97 ± 26.40R2310.74 ± 95.76PQ22,061.78 ± 819.36QR
2610,185.75 ± 257.48R23,026.64 ± 620.66X240.42 ± 10.62K5.41 ± 0.57D-H0.47 ± 0.03AB3414.3 ± 90.05P70.51 ± 2.29B-H510.19 ± 9.12E-G37,453.7 ± 927.2W
274356.59 ± 95.73O2446.97 ± 29.44C15.49 ± 1.39AB1.08 ± 0.17A9.21 ± 1.25C-H306.57 ± 10.02BC21.45 ± 0.6A-C174.01 ± 3.42A-C7331.37 ± 125.6EF
281395.66 ± 27.59GH8532.77 ± 182.94K941.11 ± 43.58T13.42 ± 0.41lMN15.45 ± 2.45GH1008.44 ± 20.59HIJ215.08 ± 8.93JK369.49 ± 21.95C-E12,491.42 ± 280.55K
29489.47 ± 9.08DE6278.88 ± 54.44H-J1240.81 ± 21.58V11.2 ± 0.81KL5.24 ± 0.65A-E2180.63 ± 94.1M112.39 ± 0.96G-I3089.58 ± 115.3RS13,408.2 ± 184.56L
30255.83 ± 11.73A-D3853.9 ± 24.7F619.18 ± 23.47P10.9 ± 1.15KL71.37 ± 1.69M1179.72 ± 17.94J802.45 ± 24.23S2944.82 ± 114.14R9738.15 ± 97.86I
31257.55 ± 8.54A-D2780.93 ± 77.85CD561.96 ± 10.64O47.77 ± 5.06T56.59 ± 2.96L1514.37 ± 95.45K658.95 ± 42.59R2141.2 ± 140.05OP8019.32 ± 348.92F-H
32544.53 ± 6.47E3671.15 ± 20.04F170.87 ± 12.02I13.03 ± 0.67LM0.29 ± 0.00A858.07 ± 43.68F-H17.74 ± 1.92AB1995 ± 80.33O7270.68 ± 126.13EF
331074.06 ± 29.14F4647.27 ± 136.25GH207.8 ± 40.27JK6.08 ± 0.44E-I2.44 ± 0.55A-C1466.53 ± 33.59K44.76 ± 0.61A-F774.18 ± 36.47H-K8223.13 ± 263.56GH
34466.79 ± 1.68C-E3113.24 ± 68.28DE169.68 ± 7.9I7.85 ± 0.36HIJ15.06 ± 2.73GH1026.78 ± 23.48H-J111.86 ± 4.39G-I2335.53 ± 123.13PQ7246.78 ± 214.09EF
3551.05 ± 1.94A305.54 ± 12.68A39.96 ± 6.33BCD7.73 ± 0.53HIJ2.13 ± 0.27A-C891.29 ± 31.8GH36.19 ± 0.89A-E3054.82 ± 161.62RS4388.7 ± 197.89C
36194.61 ± 8.14A-C1402.18 ± 28.1B95.62 ± 7.09FG4.68 ± 0.45C-G9.22 ± 1.13C-G477.73 ± 14.12C-E87.78 ± 1.76D-H1759.17 ± 53.61N4030.99 ± 61.51CD
37126.23 ± 1.92AB1419.5 ± 21.17B177.16 ± 3.65IJ6.94 ± 0.99GHIJ3.51 ± 0.39A-E697.43 ± 29.58E-G94.04 ± 2.96E-H1280.23 ± 46.13M3805.05 ± 83.11C
38592.17 ± 12.37E6582.48 ± 116.76IJ753.42 ± 15.53Q3.59 ± 0.46A-E14.2 ± 1.19FG1241.58 ± 60.26J352.78 ± 3.66O263.48 ± 10.36A-D9803.7 ± 116.59I
39381.38 ± 9.82B-E2785.14 ± 49.21CD300.07 ± 6.27L3.06 ± 0.25A-D77.62 ± 3.87N1136.92 ± 10.75IJ526.19 ± 20.32Q3186.07 ± 86.65S8396.45 ± 101.55H
401791.19 ± 29.09I11,617.64 ± 110.72O391.31 ± 23.16M4.02 ± 0.33B-F3.01 ± 0.22A-D3232.81 ± 41.13OP103.98 ± 5.05F-H944.95 ± 13.14KL18,088.92 ± 172.28O
4170.72 ± 3.11A1309.85 ± 21.19B144.92 ± 14.72HI2.47 ± 0.49A-C5.19 ± 0.53A-E443.64 ± 11.57CD294.7 ± 7.18MN139.05 ± 6.11AB2410.54 ± 48.13B
4215.71 ± 1.75A210.42 ± 2.84A69.41 ± 14.23D-F6.01 ± 0.29E-I6.5 ± 0.99A-E161.18 ± 2.06AB31.09 ± 1.34A-D120.81 ± 7.66A621.15 ± 10.07A
4333.68 ± 2.34A216.5 ± 2.46A58.56 ± 5.03C-E1.97 ± 0.17AB9.97 ± 0.79D-G145.54 ± 6.6AB123.9 ± 6.94HI207.89 ± 11.57A-D798.00 ± 14.86A
4427.63 ± 4.55A282.4 ± 5.17A26.03 ± 3.18ABC1.04 ± 0.26A4.36 ± 0.42A-E181.09 ± 5.68AB67.08 ± 3.94B-H326.18 ± 20.08A-E915.81 ± 20.17A
45105.54 ± 2.09AB0.04 ± 0.01A42.05 ± 0.57B-D1.16 ± 0.27AND302.73 ± 6.55BC0.04 ± 0.01A856.47 ± 49.15J-L1308.02 ± 43.14A
4621.56 ± 0.84A67.31 ± 1.05A88.33 ± 8.34E-G8.13 ± 0.39IJ1.53 ± 0.21AB291.44 ± 12.69BC20.23 ± 2.05A-C350.9 ± 25.66B-E849.43 ± 30.04A
4735.79 ± 1.8A1.68 ± 0.06A0.32 ± 0.08A11.25 ± 0.48KLND681.72 ± 11.04E-G0.05 ± 0.01A121.37 ± 8.89A852.18 ± 21.09A
48233.33 ± 5.72A-D946.67 ± 19.13B801.03 ± 16.99R24.5 ± 0.91QND1610.44 ± 50.06KL2510.9 ± 94.18X3547.13 ± 191.53T9673.98 ± 326.32I
Values are mean ± standard deviation of biological triplicates. Different letters between rows indicate statistically significant differences at p < 0.05. S/No corresponds to the genetic resources described in Table 1.
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Rhee, J.-H.; Choi, S.; Lee, J.-E.; Hur, O.-S.; Ro, N.-Y.; Hwang, A.-J.; Ko, H.-C.; Chung, Y.-J.; Noh, J.-J.; Assefa, A.D. Glucosinolate Content in Brassica Genetic Resources and Their Distribution Pattern within and between Inner, Middle, and Outer Leaves. Plants 2020, 9, 1421. https://doi.org/10.3390/plants9111421

AMA Style

Rhee J-H, Choi S, Lee J-E, Hur O-S, Ro N-Y, Hwang A-J, Ko H-C, Chung Y-J, Noh J-J, Assefa AD. Glucosinolate Content in Brassica Genetic Resources and Their Distribution Pattern within and between Inner, Middle, and Outer Leaves. Plants. 2020; 9(11):1421. https://doi.org/10.3390/plants9111421

Chicago/Turabian Style

Rhee, Ju-Hee, Susanna Choi, Jae-Eun Lee, On-Sook Hur, Na-Young Ro, Ae-Jin Hwang, Ho-Cheol Ko, Yun-Jo Chung, Jae-Jong Noh, and Awraris Derbie Assefa. 2020. "Glucosinolate Content in Brassica Genetic Resources and Their Distribution Pattern within and between Inner, Middle, and Outer Leaves" Plants 9, no. 11: 1421. https://doi.org/10.3390/plants9111421

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