Determination of glucosinolate contents in Brassica germplasm collections and inter- & intra-leaves distribution pattern using UPLC-MS/MS Multiple Reaction Monitoring scan mode

Intact glucosinolate (GSL) profile (five aliphatic; three aromatic, and one indolic glucosinolate) in the leaves of 50 germplasm collections and commercial cultivars of Brassica rapa, Brassica juncea, and Brassica oleracea collected from six different countries and grown under uniform cultural conditions were compared by UPLC-MS/MS. Total GSLs content ranged from 36.80 to 2383.12 μmol/kg DW. Aliphatic GSLs predominated among the entire samples representing from 23.0 to 98.9% of the total GSLs content, where gluconapin and glucobrassicanapin contributed the greatest proportion. Other GSLs such as, progoitrin (PRO), glucotropaeolin (TRO), and glucobarbarin (BAR) were found in relatively low concentrations. Principal Component Analysis (PCA) yielded three principal components with eigenvalue ≥ 1, representing 70.33% of the total variation across the entire data set. Accessions IT260822 & IT32750, and commercial cultivar, “Hangamssam2”, were well distinguished from other samples in the PCA plot due to their significantly high amount of BAR, glucobrassicin (GBC), and glucoerucin (ERU), respectively. The inter- and intra-leaf variations of GSLs were examined in three kimichi cabbage varieties. The GSLs content varied significantly among leaves in different positions (outer, middle, and inner) and sections within the leaves (top, middle, bottom, green/red, and white). Higher GSL contents were observed in the proximal half & white section of the leaves and inner positions (younger leaves) in most of the samples. GBC, gluconasturtiin (NAS), and glucoberteroin (BER) should be studied profusely in Brassica plants as some of their degradation products of GBC and NAS are useful in cancer chemopreventive functions, whereas BER takes part in the process of suppressing aging of the skin. GSLs are regarded as allelochemicals; hence, the data related to the patterns of GSLs within the leaf and between leaves at different position could be useful to understand the defense mechanism of Brassica plants. The observed variability could be useful for breeders to develop Brassica crops with high GSL content or specific profiles of GSLs as required.


Introduction
growth was maintained using nutrient solution throughout the growing season. 115 To study the glucosinolate spatial distribution within sections of the leaf of kimichi cabbage and 116 between leaves, two green pigmented ("Hangamssam" and "Alchandul") and one red pigmented ("Bbalgang 117 3-ho") commercial cultivars were selected. The inner, middle, and outer leaves were separated. Each leaf 118 was then dissected into top, middle, bottom, green/red, and white part as required. Sampling positions of 119 kimichi cabbage plant are done as shown in Fig. 1.  Sample pre-treatment, extraction and analysis of glucosinolates 127 Samples were harvested, placed in vinyl freezer bag and kept at -80 o C until further processing. The 128 frozen samples were subsequently lyophilized for 48 h using LP500 vacuum freeze-drier (Ilshin biobase Co., 129 Seoul, Korea). The freeze-dried samples were then ground to a fine powder using a mortar and pestle, and 130 held at -80ºC until analysis. 0.1 gram of lyophilized sample was mixed with 1 mL of 80% methanol in a 2 131 mL Eppendorf tube, and sonicated in ultra-sonication bath for 10 min at 30 o C. The mixture was centrifuged 132 using VS-180CFi centrifuge (Vision Scientific Co., Daejeon, Korea) (centrifuge conditions set at: 14000 rpm, 133 4 o C, and 10 min). The supernatant was transferred into a vial and glucosinolates were analyzed immediately 134 using UPLC-MS/MS. 135 The GSLs were analyzed using an Acquity UPLC System (Waters, Milford, MA, USA) coupled to 136 Xevo™ TQ-S system (Waters, MS Technologies, Manchester, UK). Chromatographic separation was carried 137 out using Acquity UPLC BEH C18 (1.7μm, 2.1 × 100mm) column (Waters Corp., Manchester, UK). The 138 flow rate was kept at 0.25 mL/min; the column temperature was maintained at 35°C; and the injection 139 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. Elution conditions were as follows: Initial condition set at 141 100% of A; 0.0 -1.0 mins, 100 to 95% of A; 1.0 -4.0 mins, 95 to 0 % A; 4.0 -4.5 mins, 0 % of A; 4.5 -5.0 142 mins, 0 to 100% of A; 5.0 -10.0 mins, 100% of A. The mass spectrometry instrument was operated in 143 negative ion electrospray ionization (ESI -) mode and Multiple reaction Monitoring (MRM). Data acquisition 144 was performed using MassLynx 4.1 software. For MS/MS detection, the ionization source parameters were 145 set as follows: capillary voltage was 3kV; the ion source and the desolvation temperatures were set as 150°C 146 350°C, respectively. The cone gas (nitrogen) and desolvation gas (also nitrogen) were set at flow rates of 150 147 and 650 L/h, respectively. Other MRM conditions are presented in Table 1  The concentrations of GSLs were also evaluated in various leaf sections and positions of two green 168 ("Hangamssam" and "Alchandul") and a red ("Bbalgang 3-ho") pigmented commercial Brassica varieties.

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The detection and quantification conditions of the GSLs by LC-MS/MS are presented in Table 1.

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Information about the germplasm collections and commercial cultivars are presented as a supplementary file 174 (S1 Table). The results of this study, which are to be presented and discussed in detail in the next sections,

Variation in GSL content between germplasm collections
As can be seen in   Table). Scores   Table 2 and S1 Table. Table 3. The white sections of the leaf contained higher total sum of 230 glucosinolates (1.16 to 24.28-fold higher) than the green/red section except in the outer leaf of "Bbalgang 3-231 ho" where the red section contained 2.8-fold greater total sum of GSLs concentration than the white section.

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The trend in total GSLs content in different sections of the leaf (top, middle, and bottom) was not strictly 233 consistent. However, in most cases higher GSLs content were observed at the proximal half of the leaves. In   Helicoverpa armigera. These authors found out that the glucosinolate abundance in the inner vs. the 282 peripheral part of the leaf affected insect feeding preference and anti-herbivore defenses. As stated in 283 previous section and  to the pattern of herbivory by insects is recommended.