| Glucosinolate and its hydrolysis products have been known as important functional components in anticancer activity with sulforaphane being the most effective one. Containing abundance of glucoraphanin and its metabolite sulforaphane, broccoli sprouts has been used as a modal vegetable to investigate the metabolism of glucosinolates. The biosynthetic pathway of glucosinolate has been elucidated in Arabidopsis thaliana. The content of glucosinolate in plant under particular situation is not only influenced by its developmental stage, but also regulated by the internal signallings as well as the external environment changes including abiotic and biotic factors. The present study investigated the role of the chemical regulations including sugars, plant hormones as well as NaCl in enhancing glucosinolates and the healthy quality of broccoli sprouts. The mutants and transgenic plants related to the signal transduction and biosynthesis of phytohormones, glucose and glucosinolates in modal plant Arabidopsis thaliana were used to clarify the mechanism underlying the regulation of glucosinolate by these chemical regulations. Following are the main results.1. Application of sucrose could induce the accumulation of glucosinolate and anthosyanins in different mechanisms in broccoli sprouts. The effect of different kinds of sugars including sucrose, glucose, mannitol and fructose on anthocyanins and glucosinolates was investigated in broccoli spouts. The results showed that all the sugars induced the accumulation of anthocyanins and glucosinolates with sucrose being the most effective one. Different regulation mechanisms of sucrose on the accumulation of glucosinolate and anthocyanin exist in broccoli sprouts. The enhancing effect of sucrose on the content of glucosinolate can be mimicked by the combined treatment of glucose and fructose with the ratio of1:1, while it can not when it refers to the anthocyanin. So it was suggested that sucrose itself can be a regulator of anthocyanin biosynthesis, and it has to be degraded into monose such as glucose to regulate the accumulation of glucosinolate. What’s more, the expression of biosynthetic genes and transcription factors of anthocyanin as well as the biosynthetic gene of glucosinolate was increased significantly by the application of sucrose. In addition, the activity of myrosinase in broccoli sprouts was affected by different kinds of sugars. Of all the sugars tested, fructose/glucose and glucose treatment significantly decreased the activity of myrosinase in broccoli sprouts. However, the sucrose, fructose and mannitol maintained the activity of myrosinase in broccoli sprouts. Thus the application of sucrose could be a useful way to improve the nutrition value of broccoli sprouts by enhancing the biosynthesis of anthocyanin and glucosinolate and maintaining the activity of myrosinase.2. The effect of JA (jasmonic acid) on the biosynthesis of glucosinolate was investigated in Arabidopsis thaliana. JA induced the accumulation of aliphatic and indolic glucosinolates significantly. However, the defective of MYB29, MYB34and MYB51dramatically weakened the enhancing effects of JA. In addition, the inducing effect of glucosinolate accumulation by JA in med25was also weakened when compared with its wild type, and no significant change in the expression of MYB34, MYB76and MYB122was observed in med25in response to JA, while all these three genes were significantly up-regulated by JA in wild type. Therefore, MED might be involved in the regulation of JA-induced glucosinolate accumulation through MYB transcription factors. The interplay of plant hormones and glucose in regulation of glucosinolate accumulation in Arabidopsis thaliana was also investigated. Glucose-induced glucosinolate biosynthesis was enhanced significantly by the addition of JA. The expression pattern of genes involved in glucosinolate biosynthetic pathway was analyzed in the seedlings treated with JA and/or glucose. The results showed that the expression level of transcription factor genes, MYB28, MYB29and MYB122as well as the eight glucosinolate biosynthetic genes tested was significantly elevated after JA and glucose treatment compared with JA or glucose treatment alone. The induction of aliphatic and indolic glucosinolate in JA-insensitive mutants, coil, jar1andjin1was less significant after treated with JA and glucose together compared with that in their corresponding wild types. Moreover, the effect of JA and glucose on glucosinolate content was dramatically reduced in glucose-insensitive mutants, rgsl-2and abi5-7. These results indicate a crosstalk between JA and glucose signalling in regulation of glucosinolate biosynthesis. JA signaling as well as RGS1, the putative membrane receptor of glucose signaling, and ABI5are involved in the synergistic effect of JA and glucose on glucosinolate accumulation.3. MED and MYB are both involved in the regulation of JA on glucosinolate biosynthesis and JA can regulate the accumulation of glucosinolate synergetically with glucose. Both treatments of NaCl alone or combined with EBR could induce the accumulation of glucosinolate in broccoli sprouts. To understand the regulation mechanism of NaCl on glucosinolate metabolism in broccoli sprouts, the contents of glucosinolates and sulforaphane, as well as myrosinase activity of broccoli sprouts germinated under0mM,20mM,40mM,60mM,80mM, and100mM of NaCl were investigated. Treatment of NaCl exerts a concentration-dependent effect on glucosinolate-myrosinase system in broccoli sprouts. NaCl treatments at relatively low concentrations (20mM and40mM) enhanced the growth of the broccoli sprouts. NaCl treatment at the concentration of60mM for5d maintained higher biomass and comparatively higher content of glucosinolates in broccoli sprouts with decreased myrosinase activity. The application of high concentration, especially100mM of NaCl, for3d endowed broccoli sprouts with abundant glucosinolates and sulforaphane. The100mM NaCl treatment significantly increased the sulforaphane content in7d old broccoli sprouts as compared to the control. What’s more, the combination of24-epibrassinolide (EBR) and NaCl also enhanced the accumulation of glucosinolates in broccoli sprouts. The content of total glucosinolates in broccoli sprouts was increased by86%after treatments with0.001mg/L EBR and40mM NaCl, while0.01mg/L EBR alone or combined with application of NaCl enhanced the content of ascorbic acid with significant increase in fresh weight of broccoli sprouts. These results indicated that low level of EBR alone or combined with NaCl treatments could be useful ways to enhance the health-promoting compounds in broccoli sprouts.4. BR inhibited the biosynthesis of glucosinolate in Arabidopsis via the transcriptional factors BZR1and BES1. The effect of EBR on glucosinolate biosynthesis in Arabidopsis thaliana was investigated by using mutants and transgenic plants involved in brassinosteroid (BR) biosynthesis and signal transduction, as well as glucosinolate biosynthesis. The results showed that EBR significantly decreased the contents of major aliphatic glucosinolates including glucoiberin (S3), glucoraphanin (S4), and glucoerucin (T4), as well as the indolic glucosinolates glucobrassicin (IM) and neoglucobrassicin (1IM). In addition, a significantly higher level of glucosinolates accumulated in the BR-deficient mutant cpd and a dramaticaly lower glucosinolate content in the transgenic plant DWF4-ox overexpressing the BR biosynthetic gene DWF4compared with their related wild-types, confirmed the repressing effect of BR on glucosinolate biosynthesis. BRI1, the receptor of BR signal transduction, was involved in regulation of glucosinolate biosynthesis by BR. Furthermore, the observation of reduced content of glucosinolates and lower expression levels of glucosinolate biosynthetic genes in35S-BZR1/bzr1-1D and bes1-D plants compared with the corresponding wild-types suggested that BZR1and BES1, two important components in BR signal transduction, are responsible for the inhibiting role of BR in glucosinolate biosynthesis. The disappearance of the repressing effect of BR on glucosinolate content in the myb28, myb34and myb122mutants indicated that these three MYB factors are important for the regulation of BR in glucosinolate biosynthesis. |
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