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

Memristor MOS Content Addressable Memory (MCAM): Hybrid Architecture for Future High Performance Search Engines

Large-capacity Content Addressable Memory (CAM) is a key element in a wide variety of applications. The inevitable complexities of scaling MOS transistors introduce a major challenge in the realization of such systems. Convergence of disparate technologies, which are compatible with CMOS processing, may allow extension of Moore's Law for a few more years. This paper provides a new approach towards the design and modeling of Memristor (Memory resistor) based Content Addressable Memory (MCAM) using a combination of memristor MOS devices to form the core of a memory/compare logic cell that forms the building block of the CAM architecture. The non-volatile characteristic and the nanoscale geometry together with compatibility of the memristor with CMOS processing technology increases the packing density, provides for new approaches towards power management through disabling CAM blocks without loss of stored data, reduces power dissipation, and has scope for speed improvement as the technology matures.Comment: 10 pages, 11 figure

Health-Promoting Phytochemicals from 11 Mustard Cultivars at Baby Leaf and Mature Stages

Mustard is a Brassica vegetable that provides a number of phytonutrients. However, the phytonutrient profile of mustard has been relatively limited. We analyzed the glucosinolates and their hydrolysis products, carotenoids, total anthocyanin and phenolic contents, and antioxidant capacity of the leaves of 11 mustard cultivars grown in a greenhouse at the baby leaf and mature stages. An aliphatic glucosinolate sinigrin and its hydrolysis products allyl isothiocyanate and 1-cyano-2,3-epithiopropane were the major phytonutrients in the mustard leaves. Carotenoids β-carotene, lutein, violaxanthin, and neoxanthin were detected. We found phytonutrient concentration and their change with plant growth were cultivar-dependent. The %RDA value for vitamin A calculated using β-carotene content and retinol activity equivalents suggests that mustard cultivars used in this study can be a good source of vitamin A. Phenolic contents and antioxidant capacity also varied among cultivars and between physiological stages. Our results suggest that mustard leaves are rich in various phytochemicals and their composition depends on cultivar and the physiological stage. This is the first report on phytochemical composition in various mustard cultivars at different physiological stages

Chemopreventive glucosinolate accumulation in various broccoli and collard tissues: Microfluidic-based targeted transcriptomics for by-product valorization

Floret, leaf, and root tissues were harvested from broccoli and collard cultivars and extracted to determine their glucosinolate and hydrolysis product profiles using high performance liquid chromatography and gas chromotography. Quinone reductase inducing bioactivity, an estimate of anti-cancer chemopreventive potential, of the extracts was measured using a hepa1c1c7 murine cell line. Extracts from root tissues were significantly different from other tissues and contained high levels of gluconasturtiin and glucoerucin. Targeted gene expression analysis on glucosinolate biosynthesis revealed that broccoli root tissue has elevated gene expression of AOP2 and low expression of FMOGS-OX homologs, essentially the opposite of what was observed in broccoli florets, which accumulated high levels of glucoraphanin. Broccoli floret tissue has significantly higher nitrile formation (%) and epithionitrile specifier protein gene expression than other tissues. This study provides basic information of the glucosinolate metabolome and transcriptome for various tissues of Brassica oleracea that maybe utilized as potential byproducts for the nutraceutical market

Glucosinolates, Carotenoids, and Vitamins E and K Variation from Selected Kale and Collard Cultivars

Glucosinolates, carotenoids, and fat-soluble vitamins E and K contents were analyzed from various kale and collard cultivars at mature stage. We found a significant difference in these phytonutrients among cultivars. Among kale cultivars, “Beira” and “Olympic Red” were the highest in the total glucosinolate and “Toscano” kale was the highest in total carotenoid content. “Scarlet” kale was highest in tocopherols. For collard, total glucosinolate was the highest in “Top Bunch” while carotenoids were the highest in “Green Glaze.” An accession PI261597 was the highest in phylloquinone. In addition to the total content of each phytonutrient class, their composition differed among cultivars, indicating that each cultivar may have differential regulatory mechanisms for biosynthesis of these phytonutrients. Our result indicates that cultivar selection may play an important role in consumption of kale and collard with greater nutritional benefit. Therefore, the result of this study will provide a more thorough profile of essential and nonessential phytonutrients of kale and collard cultivars for consumers’ choice and for future research on nutritional value of these crops

Targeted Metabolomic and Transcriptomic Analyses of “Red Russian” Kale (Brassicae napus var. pabularia) Following Methyl Jasmonate Treatment and Larval Infestation by the Cabbage Looper (Trichoplusia ni Hübner)

Methyl jasmonate (MeJA), synthesized in the jasmonic acid (JA) pathway, has been found to upregulate glucosinolate (GS) biosynthesis in plant species of the Brassicaceae family. Exogenous application of MeJA has shown to increase tissue GS concentrations and the formation of myrosinase-mediated GS hydrolysis products (GSHPs). In vitro and in vivo assays have demonstrated the potential health-promoting effects of certain GSHPs. MeJA is also known to elicit and induce genes associated with defense mechanisms to insect herbivory in Brassica species. To investigate the relationship between MeJA-induced GS biosynthesis and insect defense, three treatments were applied to “Red Russian” kale (Brassicae napus var. pabularia) seedlings: (1) a 250 μM MeJA leaf spray treatment; (2) leaf infestation with larvae of the cabbage looper (Trichoplusia ni (Hübner)); (3) control treatment (neither larval infestation nor MeJA application). Samples of leaf tissue from the three treatments were then assayed for changes in GS and GSHP concentrations, GS gene biosynthesis expression, and myrosinase activity. Major differences were observed between the three treatments in the levels of GS accumulation and GS gene expression. The insect-damaged samples showed significantly lower aliphatic GS accumulation, while both MeJA and T. ni infestation treatments induced greater accumulation of indolyl GS. The gene expression levels of CYP81F4,MYB34, and MYB122 were significantly upregulated in samples treated with MeJA and insects compared to the control group, which explained the increased indolyl GS concentration. The results suggest that the metabolic changes promoted by MeJA application and the insect herbivory response share common mechanisms of induction. This work provides potentially useful information for kale pest control and nutritional quality

Transcriptome and Metabolome Analyses of Glucosinolates in Two Broccoli Cultivars Following Jasmonate Treatment for the Induction of Glucosinolate Defense to Trichoplusia ni (Hübner)

Lepidopteran larvae growth is influenced by host plant glucosinolate (GS) concentrations, which are, in turn, influenced by the phytohormone jasmonate (JA). In order to elucidate insect resistance biomarkers to lepidopteran pests, transcriptome and metabolome analyses following JA treatments were conducted with two broccoli cultivars, Green Magic and VI-158, which have differentially induced indole GSs, neoglucobrassicin and glucobrassicin, respectively. To test these two inducible GSs on growth of cabbage looper (Trichoplusia ni), eight neonate cabbage looper larvae were placed onto each of three plants per JA treatments (0, 100, 200, 400 M) three days after treatment. After five days of feeding, weight of larvae and their survival rate was found to decrease with increasing JA concentrations in both broccoli cultivars. JA-inducible GSs were measured by high performance liquid chromatography. Neoglucobrassicin in Green Magic and glucobrassicin in VI-158 leaves were increased in a dose-dependent manner. One or both of these glucosinolates and/or their hydrolysis products showed significant inverse correlations with larval weight and survival (five days after treatment) while being positively correlated with the number of days to pupation. This implies that these two JA-inducible glucosinolates can influence the growth and survival of cabbage looper larvae. Transcriptome profiling supported the observed changes in glucosinolate and their hydrolysis product concentrations following JA treatments. Several genes related to GS metabolism differentiate the two broccoli cultivars in their pattern of transcriptional response to JA treatments. Indicative of the corresponding change in indole GS concentrations, transcripts of the transcription factor MYB122, core structure biosynthesis genes (CYP79B2, UGT74B1, SUR1, SOT16, SOT17, and SOT18), an indole glucosinolate side chain modification gene (IGMT1), and several glucosinolate hydrolysis genes (TGG1, TGG2, and ESM1) were significantly increased in Green Magic (statistically significant in most cases at 400 M) while UGT74B1 and MYB122 were significantly increased in VI-158. Therefore, these metabolite and transcript biomarker results indicate that transcriptome profiling can identify genes associated with the formation of two different indole GS and their hydrolysis products. Therefore, these metabolite and transcript biomarkers could be useful in an effective marker-assisted breeding strategy for resistance to generalist lepidopteran pests in broccoli and potentially other Brassica vegetables

Influence of 1-Methylcyclopropene Treatment on Postharvest Quality of Four Scab (Venturia inaequalis)-Resistant Apple Cultivars

Scab (Venturia inaequalis) is a very serious disease for apples causing up to 80% of loss in yield but there are only a few studies on postharvest quality of scab-resistant cultivars. In this study we evaluated the effect of 1-methylcyclopropene (1-MCP) on fruit quality, total phenolic content, and antioxidant capacity after storage of four scab-resistant cultivars and compared to a standard cultivar, “Golden Delicious.” In general, ethylene production and respiration rates significantly differed among cultivars, between control and 1-MCP-treated fruits, and between storage duration regimes. 1-MCP treatment retarded fruit softening and lowered juice pH but storage effect on soluble solids and acidity depended on cultivar and 1-MCP treatment. Total phenolic content was significantly affected by storage duration and 1-MCP treatment. Antioxidant capacity of the four scab-resistant cultivars was either similar to or significantly higher than that of “Golden Delicious” with the 1-MCP-treated fruits having significantly higher antioxidant capacity than the nontreated fruits after storage. Our results clearly show that the quality of four scab-resistant cultivars was comparable to that of “Golden Delicious” and 1-MCP effect differed among cultivars. These differences need to be considered in developing storage regime to minimize quality deterioration during long-term storage

Liquefaction of H2 molecules upon exterior surfaces of carbon nanotube bundles

We have used molecular dynamics simulations to investigate interaction of H2 molecules on the exterior surfaces of carbon nanotubes (CNTs): single and bundle types. At 80 K and 10 MPa, it is found that charge transfer occurs from a low curvature region to a high curvature region of the deformed CNT bundle, which develops charge polarization only on the deformed structure. The long-range electrostatic interactions of polarized charges on the deformed CNT bundle with hydrogen molecules are observed to induce a high local-ordering of H2 gas that results in hydrogen liquefaction. Our predicted heat of hydrogen liquefaction on the CNT bundle is 97.6 kcal kg^-1. On the other hand, hydrogen liquefaction is not observed in the CNT of a single type. This is because charge polarization is not developed on the single CNT as it is symmetrically deformed under the same pressure. Consequently, the hydrogen storage capacity on the CNT bundle is much higher due to liquefaction than that on the single CNT. Additionally, our results indicate that it would also be possible to liquefy H2 gas on a more strongly polarized CNT bundle at temperatures higher than 80 K

The theoretical study on interaction of hydrogen with single-walled boron nitride nanotubes. I. The reactive force field ReaxFFHBN development

We present a new reactive force field ReaxFFHBN derived to accurately model large molecular and condensed phase systems of H, B, and N atoms. ReaxFFHBN has been tested against quantum calculation data for B–H, B–B, and B–N bond dissociations and for H–B–H, B–N–B, and N–B–N bond angle strain energies of various molecular clusters. The accuracy of the developed ReaxFFHBN for B–N–H systems is also tested for (i) H–B and H–B bond energies as a function of out of plane in H–B(NH2)3 and H–N(BH2)3, respectively, (ii) the reaction energy for the B3N3H6+H2-->B3N3H8, and (iii) crystal properties such as lattice parameters and equations of states for the hexagonal type (h-BN) with a graphite structure and for the cubic type (c-BN) with a zinc-blende structure. For all these systems, ReaxFFHBN gives reliable results consistent with those from quantum calculations as it describes well bond breaking and formation in chemical processes and physical properties. Consequently, the molecular-dynamics simulation based on ReaxFFHBN is expected to give a good description of large systems (>2000 atoms even on the one-CPU machine) with hydrogen, boron, and nitrogen atoms