Cecil Stushnoff

ANTIOXIDANT ACTIVITY AND TOTAL PHENOLIC CONTENT OF IRANIAN OCIMUM ACCESSIONS

Basil (Ocimum basilicum L.) is used in traditional medicine, as a culinary herb and a well-known source of flavouring principles. Total antioxidant activity in 23 Iranian basil accessions was determined as Trolox equivalent antioxidant capacity (TEAC). Total phenolic contents were determined using a spectrophotometric technique, based on the Folin-Ciocalteau reagent, according to the method of Spanos and Wrolstad [Journal of Agricultural & Food Chemistry, 38 (1990) 1565] and calculated as gallic acid equivalents GAE/g dw. Total antioxidant activity varied from 10.8 to 35.7 μM Trolox, and total phenolic content ranged from 22.9 to 65.5 mg gallic acid/g dw in “Dezful I” and “Babol” accessions, respectively. A linear positive relationship existed between the antioxidant activity and total phenolic acids content of the tested basil accessions (R2=0.71). Iranian basils possess valuable antioxidant properties for culinary and possible medicinal use.

Molecular Mechanisms of Selenium Tolerance and Hyperaccumulation in Stanleya pinnata

The molecular mechanisms responsible for selenium (Se) tolerance and hyperaccumulation were studied in the Se hyperaccumulator Stanleya pinnata (Brassicaceae) by comparing it with the related secondary Se accumulator Stanleya albescens using a combination of physiological, structural, genomic, and biochemical approaches. S. pinnata accumulated 3.6-fold more Se and was tolerant to 20 μm selenate, while S. albescens suffered reduced growth, chlorosis and necrosis, impaired photosynthesis, and high levels of reactive oxygen species. Levels of ascorbic acid, glutathione, total sulfur, and nonprotein thiols were higher in S. pinnata, suggesting that Se tolerance may in part be due to increased antioxidants and up-regulated sulfur assimilation. S. pinnata had higher selenocysteine methyltransferase protein levels and, judged from liquid chromatography-mass spectrometry, mainly accumulated the free amino acid methylselenocysteine, while S. albescens accumulated mainly the free amino acid selenocystathionine. S. albescens leaf x-ray absorption near-edge structure scans mainly detected a carbon-Se-carbon compound (presumably selenocystathionine) in addition to some selenocysteine and selenate. Thus, S. albescens may accumulate more toxic forms of Se in its leaves than S. pinnata. The species also showed different leaf Se sequestration patterns: while S. albescens showed a diffuse pattern, S. pinnata sequestered Se in localized epidermal cell clusters along leaf margins and tips, concentrated inside of epidermal cells. Transcript analyses of S. pinnata showed a constitutively higher expression of genes involved in sulfur assimilation, antioxidant activities, defense, and response to (methyl)jasmonic acid, salicylic acid, or ethylene. The levels of some of these hormones were constitutively elevated in S. pinnata compared with S. albescens, and leaf Se accumulation was slightly enhanced in both species when these hormones were supplied. Thus, defense-related phytohormones may play an important signaling role in the Se hyperaccumulation of S. pinnata, perhaps by constitutively up-regulating sulfur/Se assimilation followed by methylation of selenocysteine and the targeted sequestration of methylselenocysteine.

Down-Regulating α-Galactosidase Enhances Freezing Tolerance in Transgenic Petunia

α-Galactosidase (α-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the α-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of α-Gal was modified in transgenic petunia (Petunia × hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that α-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of α-Gal mRNAs. α-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from –4°C for cold-acclimated wild-type plants to –8°C for the most tolerant antisense line. Down-regulating α-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the α-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with α-Gal provides an additional method for improving the freezing tolerance of plants.

Real-Time in Situ Monitoring of Lysozyme During Lyophilization Using Infrared Spectroscopy: Dehydration Stress in the Presence of Sucrose

AbstractPurpose. First, to investigate the role of sucrose in stabilizing protein structure (as measured by changes in the amide I band of lysozyme) caused by dehydration encountered during lyophilization. Second, to demonstrate the utility of internal reflection spectroscopy as a tool for conducting controlled lyophilization experiments. Methods. A custom-built internal reflection FTIR accessory was used to follow the entire freeze-drying process of solutions consisting of 49.4 mg/mL lysozyme in the presence and absence of 10% sucrose in real-time. Studies were carried out using D2O as a transparent medium in the infrared region of the protein amide bands. Potential self-association of the protein in the presence of sucrose was investigated using dynamic light scattering. Hydration levels were determined using a multiple regression equation. Differential scanning calorimetry (DSC) permitted characterization of the final lyophilized product. Moisture content was determined using Karl Fischer titration. Results. Throughout freezing and drying, minimal changes were observed both in frequency (1647 ± 1 cm−1) and bandwidth (46 ± 1 cm−l) of the amide I band in the presence of sucrose. In contrast, greater changes in frequency and band width were seen in the absence of sucrose. A successfully lyophilized cake was obtained which had properties of a glass as measured by DSC, with a Tg of 50°C. The lyophilized product containing sucrose had 4% moisture by weight. Three distinct rates of water desorption were discovered during drying under vacuum (50 mg/hr within the sample temperature range from −35° to −25°C; 30 mg/hr from 10° to 25°C; 1.2 mg/hr from 27° to 38°C). Conclusions. The inclusion of sucrose served to minimize perturbations of protein structure caused by freezing and dehydration stresses encountered during lyophilization (compared to studies conducted in the absence of sucrose). The results support the water replacement hypothesis and underscore the role of the sugar in preserving a native structure in the dried state. This investigation demonstrates the usefulness of infrared spectroscopy in evaluating lyophilization process parameters and formulation design.

Flavonoid profiling and transcriptome analysis reveals new gene–metabolite correlations in tubers of Solanum tuberosum L.

Anthocyanin content of potato tubers is a trait that is attracting increasing attention as the potential nutritional benefits of this class of compound become apparent. However, our understanding of potato tuber anthocyanin accumulation is not complete. The aim of this study was to use a potato microarray to investigate gene expression patterns associated with the accumulation of purple tuber anthocyanins. The advanced potato selections, CO97216-3P/PW and CO97227-2P/PW, developed by conventional breeding procedures, produced tubers with incomplete expression of tuber flesh pigmentation. This feature permits sampling pigmented and non-pigmented tissues from the same tubers, in essence, isolating the factors responsible for pigmentation from confounding genetic, environmental, and developmental effects. An examination of the transcriptome, coupled with metabolite data from purple pigmented sectors and from non-pigmented sectors of the same tuber, was undertaken to identify these genes whose expression correlated with elevated or altered polyphenol composition. Combined with a similar study using eight other conventional cultivars and advanced selections with different pigmentation, it was possible to produce a refined list of only 27 genes that were consistently differentially expressed in purple tuber tissues compared with white. Within this list are several new candidate genes that are likely to impact on tuber anthocyanin accumulation, including a gene encoding a novel single domain MYB transcription factor.

Oligosaccharides as Endogenous Cryoprotectants in Woody Plants

Cold hardy woody plant species possess remarkable capacity to acclimate and survive seasonal stresses. While growth and development are normally most active under fully hydrated conditions, plant tissues are also least adapted to cope with environmental stresses in the hydrated state. Reduction in moisture content must be accompanied by endogenous conditions which enable stabilization of cellular components. Seeds are the obvious example of such adaptation (Leopold, 1990), but many other plant organs and tissues also tolerate water loss, facilitating survival at extreme temperatures. Freeze-induced desiccation is an important step in cold acclimation and especially in achieving survival at cryogenic temperatures. Indeed, the capacity to acquire desiccation tolerance is one of the most critical stages in a series of events which permit plant tissues to stabilize and survive freezing stresses. Thus, endogenous metabolic events which accompany cold hardiness are of considerable interest.

In vitro measures used to predict anticancer activity of apple cultivars and their comparison to outcomes from a rat model of experimentally induced breast cancer.

Experiments reported herein tested the hypothesis that cultivars of apple (Malus domestica Borkh.) would demonstrate anticancer activity in vivo as predicted by in vitro measures. Freeze dried powders of Red Delicious (RD), Fuji (FJ), Golden Delicious (GD), and Granny Smith (GS) apple cultivars were evaluated. Significant differences were noted among cultivars in total phenolics (P < 0.0001), flavonoids (P < 0.0003), oxygen radical absorbance capacity (P < 0.0001), and growth inhibition in the MDA-MB-468 human breast cancer cell line relative to vehicle-treated cells (P < 0.0001). These findings were extended to predict inhibition of the postinitiation phase of 1-methyl-1-nitrosourea-induced mammary carcinogenesis by freeze-dried whole apple powders of the same cultivars. Although rats fed apple-containing diets did not have lower incidence or multiplicity of cancers than rats fed control diet, a finding consistent with epidemiological reports on fruit and breast cancer risk, differences among cultivars were noted, with the greatest difference in cancer multiplicity between GS and RD (1.46 vs. 2.47 cancers/rat; P = 0.0159). The rate of cell proliferation in mammary carcinomas differed between GS and RD (P < 0.001), whereas the apoptotic rate did not. These findings suggest altered methodology for screening apples for anticancer activity and that more diverse apple cultivars with higher phytochemical content should be evaluated.

Influence of biologically enhanced organic production on antioxidant and sensory qualities of (Malus x domestica Borkh. cv Braeburn) apples

Abstract(Malus x domestica Borkh. cv Braeburn) apples grown at Royal City, WA, USA under biologically enhanced organic (BEO) methods with nutrient and microbial enhancements were compared with apples produced using conventional methods. Apples from the outer and inner tree canopies were evaluated for antioxidant capacity, soluble solids content (SSC), shelf life, and consumer acceptability by sensory taste panels. Formazan soil tests to estimate microbial activity were correlated with fruit properties. There were no differences (P > 0.05) in 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulfonic acid]/Trolox equivalent antioxidant capacity (ABTS/TEAC) or 1,1-diphenyl-2-picrylhydrazyl radical (DPPH/TEAC) antioxidant capacity between BEO and conventional cv Braeburn apples. However, BEO apples had a higher level (P = 0.003) of total phenolics (TP) assessed by Folin–Ciocalteu reagent than conventional apples, and outer-canopy apples had higher SSC (P = 0.002), as well as higher TP, ABTS, and DPPH antioxidant properties (P < 0.01) compared to inner-canopy apples. BEO apples from both outer and inner canopies also had higher SSC (P < 0.001) than those conventionally grown. There was no difference in shelf life between BEO and conventional apples (P = 0.366), nor between outer-canopy and inner-canopy apples (P = 0.286). The overall acceptability sensory ratings for BEO apples were significantly higher (P < 0.001) than conventional fruits and outside-canopy fruits were rated superior to inner-canopy fruits (P < 0.001).