Enhancing human health promoting activity through the regulation of the methyl jasmonate mediated glucosinolate biosynthesis in Brassica oleracea
Brassica oleracea vegetables are recognized as functional foods that contain various phytochemicals such as glucosinolates (GS) and flavonoids that have health-promoting bioactivity. Recent data suggest that methyl jasmonic acid (MeJA) can increase concentrations of GS and polyphenolics in Brassica plants. In Chapter 2 tissue/organ specific responses to MeJA treatments were investigated in five cultivars of broccoli and two cultivars of kale in field plots over two years, MeJA treatments significantly increased total phenolics and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) antioxidant activity of kale leaf tissues, but had no effect on phenolics of broccoli florets. Correlation of growing degree days, precipitation per day and solar radiation with phenolic concentrations suggest that these weather related factors are associated with the enhancement of phenolics and tissue ABTS antioxidant activity. In order to evaluate if MeJA treatment can enhance induction of quinone reductase activity, an anticancer biomarker of broccoli floret extracts, MeJA treatments were applied to five broccoli cultivars in each of two years under field conditions (Chapter 3). Sulforaphane, phenethyl isothiocyanate, and hydrolysis products derived from neoglucobrassicin were significantly increased by MeJA treatment. Sulforaphane, N-methoxyindole-3-carbinol (NI3C), and neoascorbigen showed significant correlations with QR activity in hydrolysed broccoli extracts. Although sulforaphane is a known QR inducer, there is only one published report about QR activity of hydrolysis products of neoglucobrassicin (Haack et al., 2010). The concentration required for doubling specific QR activity (CD value) was calculated to be 35 and 38 µM for NI3C and neoascorbigen, respectively. The CD value of sulforaphane was previously estimated to be 0.2 µM. Given the QR inducing potency and increased amount of isothiocyanate hydrolysis product from glucoraphanin, sulforaphane is considered to be the major contributor to QR inductive activity of MeJA treated broccoli florets. Chapter 4 reports that MeJA spray treatments were applied to the kale varieties ‘Dwarf Blue Curled Vates’ and ‘Red Winter’ in replicated field plantings in 2010 and 2011, to investigate alteration of the GS composition in the harvested leaf tissue. The MeJA treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%), and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these genotypes for both season. Induction of quinone reductase (QR) activity was significantly increased by the extracts from the leaf tissue of these two cultivars. There were significant year and year by genotype interactions in the concentrations of GS and QR activity. To determine the relationship between GS hydrolysis products and QR activity, a range of concentrations of MeJA sprays were applied to kale leaf tissues of both cultivars in 2011. Correlation analysis of these results indicated that sulforaphane, NI3C, neoascorbigen, I3C, and diindolylmethane were all significantly correlated with QR activity. Thus, increased QR activity may be due to several hydrolysis products in kale leaves rather than individual products alone. MeJA treatment can also increase ethylene production, which may be harmful for the maintenance of postharvest quality of broccoli. To increase health-promoting properties of broccoli while maintaining post-harvest storage quality, 1-methylcyclopropene (1-MCP, a competitive inhibitor of plant ethylene receptor proteins) was applied to control and MeJA treated broccoli (Chapter 5). The combination of 1-MCP with MeJA treatment maximized phytochemical content and QR activity while maintaining acceptable visual quality. In order to understand the mechanisms of response in broccoli to MeJA and 1-MCP treatments gene expression of GS biosynthetic, hydrolytic, chlorophyll catabolic, and pathogen related protein (PR) genes were measured by quantitative RT-PCR. MeJA treatment significantly increased transcript abundance of the indolyl GS biosynthesis genes BoCYP79B2, BoCYP83B1, as well as myrosinase, epithiospecifier protein modifier 1 (BoESM1), and epithiospecifier protein (BoESP) genes. Consequently, neoglucobrassicin and gluconasturtiin concentrations were significantly increased by MeJA treatment. In addition, increased sulforaphane, phenethyl isothicyanate, NI3C, and neoascorbigen were significantly correlated with QR inductive activity, indicating MeJA induced GS levels enhances potential cancer chemopreventive activity. MeJA treatment significantly increased ethylene production of broccoli floret at harvest date and reduced total chlorophyll content and visual quality during post-harvest storage. 1-MCP treatment significantly suppressed mRNA levels of the chlorophyll catabolism genes, BoPaO and BoPPH. As a result, the combined treatment of MeJA and 1-MCP provides enhanced QR inductive activity while maintaining post-harvest quality compared to MeJA treatment alone. In Chapter 6, the effect of MeJA treatments were investigated in a cauliflower (B. oleracea L. var. botrytis) cultivar. Visual quality, ethylene production, GS compositional changes, and QR inductive activity of cauliflower curd extracts were examined during post-harvest storage at 4 °C. There was no significant ethylene production or visual quality loss with the MeJA treatment. Unlike broccoli, MeJA significantly increased glucoraphanin, glucobrassicin, and neoglucobrassicin, implying that GS compositional changes associated with MeJA treatment maybe species-specific. Increased GS concentrations were significantly correlated with QR inductive activity. In conclusion, MeJA treatment to cauliflower significantly enhanced QR inductive activity without a loss in post-harvest quality. Several studies to determine application protocols that maximize accumulation of GS and other phytochemicals in broccoli florets were discussed in Chapter 7. We investigated the effect of solvents and varying MeJA application concentrations, application number, and application date in days prior to harvest of broccoli florets of the cultivar ‘Green Magic’. MeJA application four days prior to harvest generated broccoli florets with the highest concentrations of GS. Although a single application of 250 µM MeJA significantly increased GS concentrations in broccoli florets, two consecutive days of treatment (four and three days prior to harvest) of 250 µM MeJA further increased total GS concentrations (primarily neoglucobrassicin) and QR activity four days prior to harvest. With increasing treatment concentrations of MeJA to broccoli florets gluconasturtiin, neoglucobrassicin, and glucoraphanin floret concentrations and QR inductive and nitric oxide production inhibitory activity were gradually increased. These application protocols were found to maximize GS concentrations and putatively enhance the health promoting properties of broccoli florets.
Brassica oleracea, Glucosinolate, Flavonoid, Quinone reductase, Antioxidant activity, Pre-harvest, Post-harvest, Methyl jasmonate (MeJa), Broccoli, Kale, Cauliflower
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