Charles R. Caldwell

Purification of Mitochondrial Glutamate Dehydrogenase from Dark-Grown Soybean Seedlings.

Proteins in extracts from cotyledons, hypocotyls, and roots of 5-d-old, dark-grown soybean (Glycine max L. Merr. cv Williams) seedlings were separated by polyacrylamide gel electrophoresis. Three isoforms of glutamate dehydrogenase (GDH) were resolved and visualized in gels stained for GDH activity. Two isoforms with high electrophoretic mobility, GDH1 and GDH2, were in protein extracts from cotyledons and a third isoform with the lowest electrophoretic mobility, GDH3, was identified in protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that GDH3 was associated with intact mitochondria. GDH3 was purified to homogeneity, as determined by native and sodium dodecyl sulfate-polyacrylamide gels. The isoenzyme was composed of a single 42-kD subunit. The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, GDH activity was 12- to 50-fold higher in the direction of reductive amination than in the direction of the oxidative deamination reaction. GDH3 had a cofactor preference for NAD(H) over NADP(H). The apparent Michaelis constant values for [alpha]-ketoglutarate, ammonium, and NADH at pH 8.0 were 3.6, 35.5, and 0.07 mM, respectively. The apparent Michaelis constant values for glutamate and NAD were 15.8 and 0.10 mM at pH 9.3, respectively. To our knowledge, this is the first biochemical and physical characterization of a purified mitochondrial NAD(H)-dependent GDH isoenzyme from soybean.

Identification of two cytosolic ascorbate peroxidase cDNAs from soybean leaves and characterization of their products by functional expression in E. coli

Abstract. Screening of a cDNA library from soybean (Glycine max (L.) Merr. cv. Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs. The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids. The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules. Escherichia coli expression systems were developed using both soybean APx cDNAs. Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products.

Estimation and Analysis of Cucumber (Cucumis sativus L.) Leaf Cellular Heat Sensitivity

Triphenyl tetrazolium chloride (TTC) reduction by cucumber (Cucumis sativus L. cv Poinsett 76 and cv Ashley) leaf discs was used as a viability assay to examine the effect of temperature pretreatment on the tissue response to acute hyperthermia. Semi-logarithmic plots of TTC reduction as a function of incubation time at different temperatures from 40 to 60[deg]C resembled the heat survival curves of animal cells. Heat inactivation rates were obtained and subjected to “quasi” Arrhenius analyses by analytical methods derived from the animal studies. The Arrhenius plots of TTC reduction rates for cv Ashley leaf discs preincubated at 25 or 37[deg]C and for cv Poinsett 76 preincubated at 37[deg]C were linear with the same activation energy (Ea) of about 80 kcal mol-1. The Arrhenius plot of cv Poinsett 76 preincubated at 25[deg]C was nonlinear with an Ea of about 80 kcal mol-1 at temperatures below 46[deg]C and an Ea of about 27.5 kcal mol-1 at temperatures above 47[deg]C. The significance of these differences is discussed in terms of the role of protein denaturation in the thermal sensitivity of cucumber disc reduction of TTC and the applicability of these methods to the analysis of plant cellular heat sensitivity.

Modification of the Cellular Heat Sensitivity of Cucumber by Growth under Supplemental Ultraviolet-B Radiation

The effect of ultraviolet B (UV-B) radiation on the thermal sensitivity of cucumber (Cucumis sativus L.) was studied using UV-B-sensitive cv Poinsett 76 and UV-B-resistant cv Ashley grown under control and elevated (300 mW m-2) UV-B radiation levels. Using both cotyledon and leaf discs, the ability of the tissue to reduce triphenyl tetrazolium chloride (TTC) was determined after treatment at 50[deg]C for various times. Semilogarithmic plots of TTC reduction as a function of time at 50[deg]C were curvilinear. They were monophasic for the control cucumber and biphasic for cucumber grown in the presence of elevated UV-B. Treatment of cucumber plants at 37[deg]C for 24 h or of tissue discs at acute UV-B levels for 1 h further modified their response to elevated temperature. These results suggest that growth of cucumber under enhanced UV-B radiation levels increased its ability to withstand elevated temperatures.

EFFECT OF ELEVATED COPPER ON PHENOLIC COMPOUNDS OF SPINACH LEAF TISSUES

ABSTRACT The effect of copper [Cu(II)] on the phenolic compounds of spinach (Spinacea oleracea L.) leaf tissues was investigated. Leaf disks (7 mm) were exposed to CuSO4·5H2O concentrations from 50 μM (12.5 ppm) to 2.5 mM (625 ppm) at pHs from 4.0 to 9.0 for 24 hr in both the light and dark. Acidified, aqueous methanol (flavonoid-rich fraction) extracts were analyzed by high performance liquid chromatography. Cu(II) altered the levels of the phenolic compounds, increasing the levels of some compounds at low Cu(II) concentrations and decreasing the levels of all the phenolic compounds at higher Cu(II) levels. In most cases, the magnitude of the Cu(II)-induced changes in spinach phenolics was independent of treatment pH. These Cu(II)-induced changes in phenolic compounds may modify plant responses to environmental or biotic stresses and alter the nutritional values of certain vegetable crops.

Effect of elevated manganese on the ultraviolet‐ and blue light‐absorbing compounds of cucumber cotyledon and leaf tissues

Abstract The effect of manganese [Mn(II)] on the pigments of cucumber (Cucumis sativus L., cv Poinsett 76) leaf and cotyledon tissues was investigated. Tissue disks (7 mm) were exposed to increasing Mn(II) concentrations from 100 μM to 2.5 mM. Acetone (carotenoid‐rich fraction) and acidified methanol (flavonoid‐rich fraction) extracts were analyzed by high performance liquid chromatography. Although none of the Mn(II)‐treated tissues showed visible damage, Mn(II) at concentrations of 250 μM and above significantly reduced (60%) the s‐carotene levels of light‐incubated leaf tissues. A major Mn(II)‐induced, UV‐absorbing compound was observed in methanol extracts of cotyledonary tissues exposed to Mn(II) in the dark. In leaf tissues, Mn(II) reduced the levels of certain UV‐absorbing compounds under both light conditions. These results demonstrate that excess leaf Mn(II) can rapidly impair isoprenoid metabolism, altering tissue carotenoid composition. Furthermore, Mn(II) may also modify phenylpropanoid metabo...

Characterization of the glutamate dehydrogenase isoenzyme system in germinating soybean

Abstract Protein extracts from different soybean (Glycine max L. Merr. cv. Williams) organs contained four glutamate dehydrogenase (GDH) isoenzymes (EC 1.4.1.2–1.4.1.4) when native polyacrylamide gels were stained for NAD-dependent activity. The four isoenzymes were designated GDH0, GDH1, GDH2 and GDH3. A combination of NAD- or NADP-specific GDH stains and immunoblot analysis of native polyacrylamide gels was used to characterize the four GDH isoenzymes. Isoenzymes GDH0, GDH1 and GDH2 demonstrated both NAD- and NADP-dependent activity, while GDH3 only had NAD-dependent activity. Based on the intensity of the different stains, GDH1 and GDH2 had greater activity with NAD than with NADP, while GDH0 had less activity with NAD than with NADP. Both GDH2 and GDH3 cross-reacted with rabbit serum raised against grape leaf NAD(H)-GDH, but the reaction was more intense with GDH3 than GDH2. Tissue print analyses were used to demonstrate the tissue-specific accumulation of GDH3 activity throughout the axis during germination in the dark. Negative controls (GDH stain solution lacking glutamate) and immunodetection with antiserum to grape NAD(H)-GDH validated the specificity of the NAD-GDH tissue print analyses. NAD-GDH activity was most abundant in the phloem of the hypocotyl hook with diffuse activity throughout the cortex. In addition, NAD-GDH activity was apparent in the epidermis of the hypocotyl hook. In the region of the hypocotyl below the hook and above the root hairs, enzyme activity was evident throughout the cortex and epidermis and absent in the vascular tissues. In the root, NAD-GDH activity was localized exclusively in the epidermal layer, particularly in the region of the functional root hairs. All other tissue types in this region of the root, including the xylem, phloem and cortex, were devoid of GDH activity. In emerging lateral roots, NAD-GDH activity was localized in the root tip and epidermis. Since only GDH3 was detected in these tissues, the data suggest that GDH3 may play a unique role in the mobilization of carbon or nitrogen in germinating seedlings.

EFFECT OF ELEVATED IRON ON ULTRAVIOLET LIGHT-ABSORBING COMPOUNDS OF CUCUMBER COTYLEDON AND LEAF TISSUES

The effect of iron [Fe(II)] on ultraviolet light-absorbing phenolic compounds of cucumber (Cucumis sativus L., cv Poinsett 76) leaf and cotyledon tissues was investigated. Tissue disks (7 mm) were exposed to increasing FeSO4 · 7H2O concentrations from 25 μ M (7 ppm) to 500 μ M (143 ppm) at pH 4.0–7.0 for 24 h in both light and dark. Acidified, aqueous methanol extracts that contain various phenolic compounds were analyzed by high performance liquid chromatography. Depending upon the treatment pH, Fe(II) altered the levels of the UV light-absorbing compounds measured at both 270 and 340 nm, increasing the levels of some compounds at low Fe(II) concentrations, which were subsequently reduced at higher Fe(II) levels. In most cases, the Fe(II)-induced change in phenolic compound concentration was dependent upon tissue type, light condition, and pH. The dose–response relations and pH dependencies for the Fe(II)-induced changes were complex. These results suggest that the FeSO4 · 7H2O treatments may specifically alter certain metabolic processes and do not simply produce reactive oxygen, which degrade the phenolic compounds.

EFFECT OF ELEVATED COPPER ON THE ULTRAVIOLET LIGHT-ABSORBING COMPOUNDS OF CUCUMBER COTYLEDON AND LEAF TISSUES

The effect of copper [Cu(II)] on the ultraviolet light-absorbing compounds of cucumber (Cucumis sativus L., cv Poinsett 76) leaf and cotyledon tissues was investigated. Tissue disks (7 mm) were exposed to CuSO4 · 5H2O concentrations from 50 μM (12.5 ppm) to 2.5 mM (625 ppm) at either pH 6.0 or 4.5 for up to 48 h in both light and dark. Acidified, aqueous methanol extracts (flavonoid-rich fraction) were analyzed by high performance liquid chromatography. Copper(II) altered the levels of the UV-absorbing compounds (340 nm), increasing the levels of some compounds at low Cu(II) concentrations and decreasing the levels of the UV-absorbing compounds at higher Cu(II) levels. In most cases, the magnitude of the Cu(II)-induced changes in cucumber phenolics was dependent upon tissue type, light condition, pH, and treatment duration. Since plant leaf phenolic compounds extracted under these conditions may have antioxidative activity, the ability to chelate prooxidative minerals and protect against ultraviolet B radiation, the Cu(II)-induced changes in phenolic compounds may modify plant responses to environmental stresses and alter the nutritional values of certain vegetable crops.

Effect of supplemental ultraviolet radiation on the concentration of phytonutrients in green and red leaf lettuce (Lactuca sativa) cultivars

Eight cultivars each of red and green leaf lettuce were raised in a greenhouse with supplemental UV radiation, either UV-A (wavelengths greater than ca. 315 nm) or UV-A+UV-B (wavelengths greater than ca. 290 nm; 6.4 kJ m-2 daily biologically effective UV-B), or no supplemental UV (controls). Several phytonutrients were analyzed in leaf flours to identify lines with large differences in composition and response to UV-B. Red leaf lettuce had higher levels of phenolic acid esters, flavonols and anthocyanins than green lines. Both green and red lines exposed to UV-B for 9 days showed 2-3-fold increases in flavonoids compared to controls, but only 45% increases in phenolic acid esters, suggesting these compounds may be regulated by different mechanisms. There were large differences between cultivars in levels of phenolic compounds under control conditions and also large differences in UV-B effects. Among red varieties, cv. Galactic was notable for high levels of phenolics and a large response to UV-B. Among green varieties, cvs. Black-Seeded Simpson and Simpson Elite had large increases in phenolics with UV-B exposure. Photosynthetic pigments were also analyzed. Green leaf lettuce had high levels of pheophytin, a chlorophyll degradation product. Total chlorophylls (including pheophytin) were much lower in green compared to red varieties. Lutein, a carotenoid, was similar for green and red lines. Total chlorophylls and lutein increased 2-fold under supplemental UV-B in green lines but decreased slightly under UV-B in red lines. Lettuce appears to be a valuable crop to use to study phytochemical-environment interactions.