Accumulating evidence indicates that there is a multiplicity of cytochrome P-450 enzymes in plants. These monooxygenases are implicated in the metabolism of sterols, terpenes, gibberellins, isoflavonoids, and xenobiotics. Evidence that cytochromes P-450 are involved in the detoxification of herbicides (chlorotoluron, primsulfuron, and diclofop) includes photoreversible CO inhibition of the reactions, and a requirement for 02 and NADPH. Several cytochromes P-450, Mr 45,000 to 65,000, have been isolated, including hydroxylases of cinnamic acid, 3,9-dihydroxypterocarpan, and digitoxin. In some cases the purified cytochrome P-450 has been successfully reconstituted with NADPH:cytochrome P-450 reductase (Mr 72,000-84,000 protein). This reductase appears to be a nonspecific electron donor to different forms of cytochrome P-450. Immunological techniques and specific inhibitors (triazoles, imidazole derivatives) are being used to characterize plant cytochromes P-450 and the NADPH:cytochrome P-450 reductase. Specific cytochromes P-450 are induced by wounding or pathogens, others are expressed in specific cell types. Plant cytochromes P-450 are found in various subcellular locations, including endoplasmic reticulum, plasma membranes, glyoxysomes, and perhaps mitochondria. A cytochrome P-450 demethylase from avocado has recently been sequenced and found to have a hydrophobic N terminus similar to the membrane anchor of cytochromes P-450 from other organisms. The existence of cytochromes P-450 in different subcellular locations suggests that there are many genes for cytochromes P-450 in plants which have yet to be identified and classified.Cyt P-450 are membrane-bound heme-containing proteins which have been implicated in a variety of oxidative reactions in plant tissues. A wide range of reactions are mediated by specific forms of these proteins. Cyt P-450-linked enzymes have been implicated in biosynthetic pathways leading to the synthesis of lignin phenolics, membrane sterols, phytoalexins, and terpenoids (33). It has been postulated that plants have evolved highly specific Cyt P-450-linked secondary pathways to produce defense-related phytoalexins, while animals have evolved parallel less specific, Cyt P-450-linked systems to detoxify ingested phytoalexins and other xenobiotics (11,22 at the expense of the 450 nm peak (25). For this reason the CO-hemoprotein adduct with the 420 nm peak, termed P-420, is often considered to be a degradation product of Cyt P-450.In addition to the nonspecific inhibition by CO, certain Cyt P-450 activities are selectively inhibited by various imidazole, pyrimidine, and triazole derivatives. Inhibitor binding also can be determined spectrophotometrically by changes in difference spectra. A typical Cyt P-450 spectrum of any mem-669 www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

Human amylin-derived oligomers and aggregates are believed to play an important role in the pathogenesis of type II diabetes mellitus (T2DM). In addition to amylin-evoked cell attrition, T2DM is often accompanied by elevated serum copper levels. Although previous studies have shown that human amylin, in the course of its aggregation, produces hydrogen peroxide (H2O2) in solution, and that this process is exacerbated in the presence of copper(II) ions (Cu2+), very little is known about the mechanism of interaction between Cu2+ and amylin in pancreatic β-cells, including its pathological significance. Hence, in this study we investigated the mechanism by which Cu2+ and human amylin catalyze formation of reactive oxygen species (ROS) in cells and in vitro, and examined the modulatory effect of Cu2+ on amylin aggregation and toxicity in pancreatic rat insulinoma (RIN-m5F) β-cells. Our results indicate that Cu2+ interacts with human and rat amylin to form metalo-peptide complexes with low aggregative and oxidative properties. Human and non-amyloidogenic rat amylin produced minute (nM) amounts of H2O2, the accumulation of which was slightly enhanced in the presence of Cu2+. In a marked contrast to human and rat amylin, and in the presence of the reducing agents glutathione and ascorbate, Cu2+ produced μM concentrations of H2O2 surpassing the amylin effect by several fold. The current study shows that human and rat amylin not only produce but also quench H2O2, and that human but not rat amylin significantly decreases the amount of H2O2 in solution produced by Cu2+ and glutathione. Similarly, human amylin was found to also decrease hydroxyl radical formation elicited by Cu2+ and glutathione. Furthermore, Cu2+ mitigated the toxic effect of human amylin by inhibiting activation of pro-apoptotic caspase-3 and stress-kinase signaling pathways in rat pancreatic insulinoma cells in part by stabilizing human amylin in its native conformational state. This sacrificial quenching of metal-catalyzed ROS by human amylin and copper’s anti-aggregative and anti-apoptotic properties suggest a novel and protective role for the copper–amylin complex.

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During the period of most active leaf expansion, the foliar dark respiration rate of soybeans (Glycine max cv Williams), grown for 2 weeks in 1000 microliters CO2 per liter air, was 1.45 milligrams CO2 evolved per hour leaf density thickness, and this was twice the rate displayed by leaves of control plants (350 microliters CO2 per liter air). There was a higher foliar nonstructural carbohydrate level (eg. sucrose and starch) in the CO2 enriched compared with CO2 normal plants. For leaves. These increases, however, are due to an increase in the volume and water content of the cells, and not an increase in the actual number of cells (15). Recently, attention has been given to the role of carbohydrate status in regulating carbon metabolism (9). It has been suggested that high carbohydrate levels inhibit photosynthesis (3, 25). Azcon-Bieto (3) postulates that above a certain critical level of carbohydrate, CO2 assimilation would decrease due to carbohydrate accumulation impairing the production or consumption of ATP/NADPH in photosynthesis. However, Robinson (22) has reported higher photosynthetic activities associated with high starch and sucrose levels.Carbohydrate status may also have an effect on dark respiration. A high carbohydrate status has been associated with elevated respiration rates in a number of studies (1,2,5,20,26). Dark respiration is thought to have two components, growth and maintenance. Growth respiration occurs in those parts of a plant which are actively dividing or expanding (21). The rate ofgrowth respiration is thought to be related to the rate of photosynthesis. Maintenance respiration occurs throughout the life of the plant and is proportional to the Dw ofthe plant (18, 21). During plant growth both types of respiration occur, but growth respiration would decrease as the leaf matures. Both are thought to involve carbohydrate oxidation through glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle (2).In this study, long-term CO2 enrichment was used to increase carbohydrate levels in order to investigate the relationship between dark respiration and carbohydrate status. The effect of increased carbohydrate status on dark respiration was studied by monitoring dark CO2 release from whole leaves. Subcellular fractionation of enriched and control leaves was performed to determine if there were increases in the activity or number of mitochondria as a consequence of a high CO2 level. MATERIALS AND METHODSPlant Material. Soybeans (Glycine max cv Williams) were planted in 15-cm pots containing vermiculite and were grown in Environmental Growth Chambers No. M-2 at 25°C, with a 12-h photoperiod. The plants were illuminated with a mixture of fluorescent and incandescent bulbs at 600 uE/m2 at pot height. They were flushed daily with a nutrient solution described previously (22

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