The process of CaCl2-HCl electrolyzed water’s production and broccoli sprouts’ cultivation is shown in Fig. 1. Details can be seen in “MATERIALS AND METHODS”.
Change of calcium, ROS and MDA contents in broccoli sprouts
As shown in Fig. 2a, the calcium content of broccoli sprouts gradually increased during the growth. Compared to tap water, CHEW significantly improved the level of calcium of broccoli sprouts. On the 2nd, 4th, 6th and 8th day of the growth, compared to tap water, calcium content of broccoli sprouts under CHEW treated were significantly increased by11.3%, 22.9%, 52.8% and 50.2% respectively. It indicated that CHEW treatment further promotes calcium accumulation in broccoli sprouts during the growth.
To investigate the effects of CHEW on the physiological changes in broccoli sprouts, we found that the H2O2 content of broccoli sprouts gradually decreased during the growth, while the O2− and MDA contents first increased and then decreased (Fig. 2b-d). In plants, ROS are key signaling molecules in abiotic and biotic stress sensing that enable cells to rapidly respond to different stimulus and establish defense mechanisms 21. MDA can reflect the broccoli sprouts response to oxidative stress. Compared to tap water, CHEW treated reduced contents of ROS and MDA significantly of broccoli sprouts during the growth. On the second day of the growth, compared to tap water, contents of H2O2, O2− and MDA in broccoli sprouts under CHEW treatment were significantly decreased by 8.8%, 25.7% and 48.8% respectively. The results indicated that CHEW treatment reduced ROS levels in broccoli sprouts during the growth and alleviated membrane lipid peroxidation induced by tap water.
Glucosinolate biosynthesis of broccoli sprouts
The species of glucosinolates identified of broccoli sprouts are listed in supplementary Figure S1. There are 3 aliphatic glucosinolates (glucoraphanin, glucoerucin, glucoalyssin) and 4 indole glucosinolates (4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, glucobrassicin, neoglucobrassicin) in broccoli sprouts. As shown in Table 1, glucoraphanin and glucoerucin are the main glucosinolates of broccoli sprouts. Besides, glucoraphanin is produced by desulfurization of glucoerucin and can be further converted to gluconapin (too low to be detected in this study). During the growth, total glucosinolates’ content of broccoli sprouts increased and then decreased. On 4th day, the contents of glucoraphanin and total glucosinolates reached the maximum values of 120.38 µmol/g DW and 176.46 µmol/g DW, respectively. This may be due to the fact that seed germination activates a number of physiological activities such as glucosinolate biosynthesis in plants, resulting in an increase in the content of glucosinolate in broccoli sprouts 2. Besides, with the extension of growth time, glucosinolates in broccoli sprouts were gradually decomposed and used to synthesize other sulfur-containing substances, so that the content of glucosinolates gradually decreased 22. Under CHEW treated, the glucosinolates’ content in broccoli sprouts during growth was always significantly higher than that of tap water treatment. On 4th day, contents of glucoraphanin and total glucosinolates in broccoli sprouts with CHEW treated reached maximum values of 141.24 µmol/g DW and 195.50 µmol/g DW respectively and were significantly increased by 17.32% and 10.79% respectively compared to tap water. The results indicated that CHEW could change the glucosinolate biosynthesis of broccoli sprouts during the growth and further increase the glucosinolate content.
Table 1
The total and individual glucosinolate contents (µmol/g DW) in broccoli sprouts under different treatments
Groups | Aliphatic glucosinolate | Indole glucosinolates | Total glucosinolates |
GRA | GER | Aly | 4OHGBS | 4MOGBS | GBS | nGBS |
2d | TW | 110.08 ± 1.96ef | 38.47 ± 0.17d | 2.59 ± 0.01b | 2.95 ± 0.08b | 0.42 ± 0.01f | 1.31 ± 0.01e | 0.51 ± 0.01f | 156.34 ± 1.87e |
CHEW | 115.84 ± 2.18cd | 38.61 ± 0.13cd | 2.59 ± 0.13b | 2.71 ± 0.08b | 0.35 ± 0.01f | 1.27 ± 0.01e | 0.45 ± 0.01f | 161.83 ± 2.34d |
4d | TW | 120.38 ± 1.89bc | 45.16 ± 0.28a | 2.71 ± 0.03ab | 3.47 ± 0.02a | 0.78 ± 0.01e | 2.39 ± 0.05d | 1.57 ± 0.04e | 176.46 ± 1.69b |
CHEW | 141.24 ± 0.57a | 43.31 ± 0.50b | 2.79 ± 0.04a | 2.78 ± 0.01b | 0.91 ± 0.02d | 2.49 ± 0.01cd | 1.97 ± 0.04d | 195.50 ± 1.05a |
6d | TW | 105.52 ± 1.02fg | 33.85 ± 1.46e | 1.89 ± 0.13e | 2.06 ± 0.18c | 1.50 ± 0.10c | 2.52 ± 0.08c | 2.33 ± 0.30c | 150.16 ± 0.94f |
CHEW | 122.01 ± 1.29b | 37.30 ± 0.46d | 2.11 ± 0.02d | 2.14 ± 0.03c | 1.51 ± 0.03c | 2.64 ± 0.12b | 2.60 ± 0.13b | 170.80 ± 1.03c |
8d | TW | 103.43 ± 1.58g | 37.23 ± 0.22d | 2.21 ± 0.02cd | 2.05 ± 0.05c | 2.14 ± 0.01b | 2.53 ± 0.03c | 2.74 ± 0.06b | 152.35 ± 1.54ef |
CHEW | 114.93 ± 1.92de | 40.37 ± 0.19c | 2.30 ± 0.06c | 1.95 ± 0.05c | 2.50 ± 0.04a | 3.27 ± 0.08a | 3.20 ± 0.06a | 168.54 ± 1.96c |
TW: tap water; CHEW: CaCl2-HCl electrolyzed water. GRA, glucoraphanin; GER, glucoerucin; Aly, glucoalyssin; 4OHGBS, 4-hydroxyglucobrassicin; 4MOGBS, 4-methoxyglucobrassicin; GBS, glucobrassicin; nGBS, neoglucobrassicin. Data are expressed as the mean ± standard deviation (SD), and values were obtained by three replicate measurements. Different letters indicate significant differences (P < 0.05) with a column. |
Glucosinolate hydrolysis of broccoli sprouts
According to TIC map of glucosinolates’ hydrolyzates, we found that 5 hydrolysis products were detected in broccoli sprouts (Fig. 3a). After further identified, they were 3 isothiocyanates (sulforaphane, 4-isothiocyanato-1-buene, erucin) and 2 nitriles (sulforaphane nitrile and erucin nitrile) respectively (Table 2). The content of sulforaphane in isothiocyanates is the highest, followed by erucin. The hydrolysis products of glucoraphanin are sulforephane and sulforephane nitrile; erucin and erucin nitrile are the hydrolysis products of glucoerucin; 4-isothiocyanato-1-buene is generated by gluconapin hydrolysis.
Table 2
GC-MS analysis result of the glucosinolate’ hydrolyzates in broccoli sprouts
NO. | Compound | CAS | Retention time (min) | Formula |
1 | 4-isothiocyanato-1-butene | 3386-97-8 | 5.759 | C5H7NS |
2 | Erucin nitrile | 59121-25-4 | 9.202 | C6H11NS |
3 | Erucin | 4430-36-8 | 12.437 | C6H11NS2 |
4 | Sulforaphane nitrile | 61121-66-2 | 13.599 | C6H11NOS |
5 | Sulforaphane | 4478-93-7 | 16.443 | C6H11NOS2 |
As shown in Fig. 3b-f, contents of isothiocyanates gradually increased during the growth of broccoli sprouts, and the content of nitriles first increased and then decreased. On the 8th day, the contents of sulforaphane, erucin and 4-isothiocyanato-1-buene of broccoli sprouts under CHEW treatment reached the maximum values of 34.78 mg/g DW, 5.43 mg/g DW and 59.65 ug/g DW, respectively. Compared to tap water, contents of three isothiocyanates of broccoli sprouts under CHEW treated on 8th day were significantly increased by 45.93%, 34.04% and 185.15%, respectively. CHEW increased the isothiocyanates’ content significantly during broccoli sprouts’ growth, while also reducing nitriles’ formation. On the 8th day, contents of sulforaphane nitrile and erucin nitrile of broccoli sprouts with CHEW treatment were significantly decreased by 18.38% and 77.08%, respectively, compared to tap water treatment. In addition, the activities of myrosinase and ESP increased gradually during broccoli sprouts’ growth (Fig. 3g, h). The activity value of myrosinase is much greater than that of ESP. On the 8th day, compared to tap water, myrosinase’ activity of broccoli sprouts under CHEW treatment was significantly increased by 52.46%. The above results indicated that CHEW treatment could affect the hydrolysis direction of glucosinolates during broccoli sprouts’ growth, and promoted the formation of isothiocyanates mainly via improving myrosinase’ activity.
Effect of CHEW on the expressions of genes related to glucosinolates metabolism
Molecular mechanism of aliphatic glucosinolates biosynthesis has been elucidated, mainly including the following three stages: (1) extension of the methionine side chain; (2) formation of the glucosinolate core structure; (3) dimeric glucosinolate side chain secondary modification 23,24. Elong and BCAT4 are the main genes involved in the first stage. The second stage is a common part of the formation of all types of glucosinolates, and is also a key step in the glucosinolates biosynthesis. CYP79F1 and CYP83A1 are the main genes involved in this stage. In the third stage, with flavin-containing monooxygenase’s (FMO) catalysis, side chain of glucoerucin undergoes oxidation to form glucoraphanin. Then glucoraphanin is catalyzed by 2-Oxoglutarate-Dependent Dioxygenases (AOP) to form gluconapin. Genes involved in this stage include FMOGS−OX1 and AOP2.
The expressions of glucosinolate biosynthesis-related genes during the growth of broccoli sprouts are shown in Fig. 4a-f The highest expression period of different genes will be different during the growth of broccoli sprouts. The relative expressions of Elong, BCAT4, CYP79F1, CYP83A1 and FMOGS−OX1 increased first and then decreased in broccoli sprouts during the growth. On the 6th day, the relative expressions of Elong, BCAT4, CYP79F1, CYP83A1, and FMOGS−OX1 in broccoli sprouts reached the maximum value. It is consistent with the changing trend of glucosinolates during broccoli sprouts’ growth (Table 1). The relative expressions of Elong, BCAT4, CYP79F1 and FMOGS−OX1 in broccoli sprouts under CHEW treatment were 2.55- fold, 3.21- fold, 1.71- fold and 1.96- fold higher respectively than those in the tap water treatment on the 6th day. The relative expression of AOP2 increased gradually in broccoli sprouts during the growth. The relative expressions of CYP83A1 and AOP2 in broccoli sprout with CHEW treatment reached the maximum on the 8th day. On the 8th day, compared to tap water, the relative expressions of CYP83A1 and AOP2 of broccoli sprouts with CHEW treatment were significantly increased by 47.27% and 113.84%, respectively. It indicated that CHEW treatment promoted glucosinolate biosynthesis by increasing the expression of glucosinolate synthesis-related genes in broccoli sprout during the growth.
It can be seen from Fig. 4g-i that the relative expression of glucosinolate hydrolysis-related genes MYR, ESP and ESM1 increased first and then decreased during the growth of broccoli sprouts. The genes related to glucosinolate hydrolysis all reached the highest expression level in broccoli sprouts on the 4th day. On the 4th day, compared to tap water, the relative expressions of MYR and ESM1 of broccoli sprouts under CHEW treatment decreased by 66.84% and 10.59% respectively, while the relative expression of ESP was 3.65- fold that of the tap water treatment. This may be due to the fact that the glucosinolate content reached maximum value on the 4th day, which trigger negative feedback regulation to promote expression of glucosinolate hydrolysis-related genes to maintain balance of glucosinolate metabolism in broccoli sprouts.
This may be because the glucosinolate content reached maximum value on the 4th day, which negatively feedback regulates the glucosinolate metabolic balance in broccoli sprouts by promoting glucosinolate hydrolysis-related genes. In addition, the change trends of the expression of MYR and ESP were inconsistent with the change trends of activities of myrosinase and ESP in Fig. 3g-h. On the one hand, myrosinase’s activity is not only regulated by the gene MYR, but also influenced by various factors such as coenzyme factor ascorbic acid, pH, metal ions, water activity and temperature 25. On the other hand, the activity of ESP is also interfered by the epithiospecifier modifier protein (ESM1)26. Above results showed CHEW mainly promoted hydrolysis of glucosinolates to generate more isothiocyanates through the negative feedback of changes in glucosinolate content during broccoli sprouts’ growth.
Localization of calcium and calcium signal transduction in broccoli sprouts
Three types of calcium are existed in broccoli sprouts’ cells: free calcium, bound calcium and stored calcium 27. The free calcium content is not high, only below 10− 6mol/L. Bound calcium is able to bind to some specific strong-affinity structural components. Organelles and cell walls usually contain high levels of stored calcium, above 10− 6 mol/L, accounting for most of the total intracellular calcium. These forms of calcium can be converted into each other in order to meet the needs of the physiological response of plant. Since the leaves of the broccoli sprouts on 2th day were too small to be detected, the calcium distribution only on 4th, 6th, 8th days were discussed. The dynamic distribution of calcium in broccoli sprouts is shown in Fig. 5a. A small amount of calcium was distributed in the cell wall and vacuole in broccoli sprouts during the growth. In plant cell walls, calcium binds to pectin in the gum layer in the cell wall to form calcium pectin. Pectin calcium can not only bond the cell wall material, but also participate in regulating membrane permeability and increasing the strength of the cell wall. In the CHEW treatment group, it was clearly observed that on the 4th day, calcium influx into the cells through endocytosis. Large amounts of calcium accumulated in the vacuole on 6th day. And on 8th day, the calcium aggregated within the vacuoles gradually dispersed. The type of calcium in the vacuoles is mainly calcium oxalate, which is formed by combining with organic acids produced by the secondary metabolism of broccoli sprouts. The formation of calcium oxalate is reversible, which can regulate intracellular ion balance and cell membrane permeability, and is affected by intracellular calcium concentration and calmodulin activity.
It can be seen from Fig. 5b that the relative expression of respiratory burst oxidase gene -RBOHD in broccoli sprouts gradually decreased during the growth. Compared to tap water group, expression of RBOHD under CHEW treatment was significantly reduced by 23% and 66% on 4th day and 8th day, respectively. On 2th day, expression of RBOHD was significantly higher in the CHEW treatment compared to tap water treatment, which may be due to the main role of the oxidative components in CHEW such as HClO and ClO−. RBOHD is an enzyme involved in ROS generation and can act as an electron donor to reduce O2 to O2−, which is then rapidly converted to H2O228,29 Calcium regulates the activity of RBOHD by binding to it and changing its conformational. The changing trend of RBOHD relative expression is consistent with the results in Fig. 1 that the CHEW reduced ROS contents significantly during broccoli sprouts’ growth. This indicated CHEW treatment reduced ROS contents in broccoli sprouts by regulating the Ca-RBOHD enzyme signaling pathway, thereby alleviating the oxidative stress caused by tap water.
The relative expression of calcium ion channel gene - CNGC increased first and then decreased during the growth of broccoli sprouts (Fig. 5c), which represented the change of the influx rate of calcium. This result is consistent with the dynamic change process of calcium observed in Fig. 2d and Fig. 4a. On 8th day, the rate of increase in calcium content of broccoli sprouts decreased from 52.8–50.2% (Fig. 2d). During the growth, the intracellular calcium concentration of broccoli sprouts gradually increased, and the mesophyll cells maintained the intracellular calcium balance by regulating the influx rate of calcium. In addition, the relative expression of calcium sensors (calmodulin, calmodulin-like protein, calcium-dependent protein kinase) genes – CaM, CML and CDPK increased first and then decreased during broccoli sprouts’ growth, and their expressions in CHEW treatment was always higher than that in tap water (Fig. 5d-f). Compared to tap water, expressions of CaM, CML and CDPK in broccoli sprouts under CHEW treatment on 2th day were significantly increased by 146%, 89% and 145%, respectively. It indicated that CHEW treatment activated the calcium signal pathway in broccoli sprouts, which was mainly transmitted downstream through the calcium sensors: CaM, CML and CDPK proteins.
It can be seen from Fig. 4G that the relative expression of IQD1 in broccoli sprouts gradually decreased during the growth. On the 4th and 6th day, relative expression of IQD1 in CHEW was significantly lower than that in tap water treatment. IQD1 is a calcium-binding nuclear protein that regulates glucosinolate metabolism by integrating intracellular calcium signal. Previous research has found that overexpression of IQD1 can promote the accumulation of aliphatic glucosinolates in Arabidopsis thaliana, while when the accumulation of aliphatic glucosinolates reaches a certain level, IQD1 will inhibit the expression of the glucosinolate biosynthesis-related gene CYP79F1 and the activity of myrosinase through negative feedback regulation 30. In this study, the trend of IQD1 expression during broccoli sprouts’ growth was consistent with changes of glucosinolate content. It indicated that calcium signal could promote glucosinolate metabolism in broccoli sprouts through IQD1.
The mechanism of CHEW treatment regulating glucosinolates biosynthesis in broccoli sprouts.
The molecular mechanism of CHEW regulating glucosinolate metabolism in broccoli sprout during the growth via calcium signal is shown in Fig. 6. Calcium of CHEW enter cells mainly through endocytosis and calcium ion channels (CNGCs) on the cell membrane to generate calcium signal. On the one hand, calcium signal regulates the activity of NADPH oxidase RBOHD by changing the conformation of it, thereby affecting the level of reactive oxygen species in broccoli sprouts and relieving the oxidative stress caused by tap water. On the other hand, calcium sensors (CaM, CML, CDPK) in broccoli sprout receive the calcium signal and transmit it to the nuclear calcium-binding protein IQD1. Previous study showed that IQD1 affected the relative expressions of glucosinolate biosynthesis-related genes and promotes glucosinolate accumulation in watercress 31. However, when the glucosinolate content in plants increased to a certain extent, IQD1 will inhibit the biosynthesis of glucosinolates through negative feedback 30. Overall, CHEW activated calcium signal in broccoli sprouts and promoted glucosinolate metabolism by increasing the relative expressions of glucosinolate biosynthesis-related genes and the negative feedback of glucosinolates.