Uniconazole inhibits stress-induced ethylene in wheat and soybean seedlings

Previous studies have shown that uniconazole inhibits ethylene synthesis and protects plants from various stresses. The present research was conducted to delineate the mechanism of ethylene inhibition by uniconazole [(E)-(p-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol]. Following heat stress of 48°C for 3 h, the shoots of the control wheat seedlings became desiccated, and the seedlings lost 23% of their fresh mass 8 h after stress. The control soybean seedlings had epinastic unifoliate leaves 5 h after foliar application (4.4 g.a.i./ha) of the herbicide triclopyr [(3,5,6-trichloro-2-pyridinyl)oxyacetic acid]. Soil drench applications of uniconazole, a potent member of the triazole family, reduced these symptoms associated with heat and herbicide stress in wheat (5.0 mg/L) and soybean (0.4 mg/L) seedlings, respectively.Basal ethylene production was inhibited 32 and 48% by uniconazole in the wheat and acotyledonous soybean seedlings, respectively. Following a 48°C heat stress, 1-aminocyclopropane-1-carboxylic acid (ACC) levels increased 40% in both the control and uniconazole-treated wheat seedlings. After triclopyr application, ACC levels increased 400% in both the control and uniconazoletreated soybean seedlings. The increased ACC levels, following stress, were accompanied by increased ethylene production from the control, but not from the uniconazole-treated wheat and acotyledonous soybean seedlings. Uniconazole treatment did not significantly change the basal or stress-induced N-malonyl-1-aminocyclopropane-1-carboxylic acid (MACC) levels compared to controls. These results suggest that uniconazole inhibits ethylene synthesis by interfering with the conversion of ACC to ethylene in wheat and acotyledonous soybean seedlings. Ethylene production and ACC conversion were not inhibited by uniconazole in excised soybean cotyledons. These results indicate that different ethylene-forming enzyme (EFE) systems operate in the soybean acotyledonous seedling and cotyledon, and the system in the former is inhibited by uniconazole.     

Modulation of ethylene synthesis in acotyledonous soybean and wheat seedlings

The characteristics of ethylene production and ACC conversion in 8-day-old soybean seedlings were examined and a relationship between cytochrome P-450 activity and ethylene-forming enzyme (EFE) activity was found. An atmosphere containing 10% carbon monoxide (CO) significantly inhibited ethylene production and ACC conversion in control soybean seedlings, but had only a slight effect on soybean seedlings treated with uniconazole. Foliar application of triclopyr, a pyridine analogue of the phenoxy herbicides, significantly increased ethylene production and ACC conversion in control, but not in uniconazoletreated seedlings. Triclopyr treatment also resulted in a three-fold increase in extractable cytochrome P-450 of 5-day-old etiolated soybeans. At equimolar concentrations tetcyclacis was more effective than uniconazole in reducing shoot elongation and endogenous ethylene production. Although uniconazole and tetcyclacis did not inhibit ACC conversion in nonherbicide-treated soybean seedlings, they did prevent the observed increase in ACC-dependent EFE activity following triclopyr application. However, the rate of ACC conversion in etiolated soybean segments was sensitive to uniconazole, and tetcyclacis inhibited the rate of ACC conversion by 2.6-fold in etiolated soybean segments within 4 h after treatment. Microsomal membranes were isolated from 5-day-old naphthalic anhydride-treated etiolated wheat shoots as this tissue contains much higher cytochrome P-450 levels than soybean shoots. Optical difference spectroscopy demonstrated that ACC generated binding spectrum characteristic of a reverse-type-I cytochrome P-450 substrate when combined with reduced microsomes. In vitro conversion of ACC to ethylene by microsomal membranes was NADPH-dependent, inhibited by CO, and had an apparent K_m and V_max of 45 M and 0.345 nl/mg protein/h, respectively. These results suggest that cytochrome P-450-mediated monooxygenase reactions may be intimately involved in the conversion of ACC to ethylene in young soybean and wheat seedlings.     

Acid-bacterial and ferric sulfate leaching of pyrite single crystals

Pyrite single-crystal cubes were cut, polished. and x-rayed to produce orientations of (100), (110), (111), and (112). These crystallographically developed surfaces then were prepared to expose an area of 1 cm(2), and the remainder of the crystal was coated with an acid-resistant silicone cement. Crystals with representative orientations then were leached in ferric sulfate solutions adjusted to a pH of 2.3 with H(2)SO(4) containing up to 6 x 10(3) ppm of Fe(3+) at 30 and 55 degrees C. Leaching was also conducted in acid-bacterial lixiviants containing Thiobacillus ferrooxidans at 30 degrees C and a thermophilic microorganism at 55 degrees C. Surface corrosion and pitting associated with pyrite leaching were examined by scanning electron microscopy. Pyrite leaching in ferric sulfate solutions was observed to be different when compared to acid-bacterial leaching. Ferric sulfate leaching required nearly 2 x 10(3) ppm of Fe(3+) at 30 degrees C while acid-bacterial leaching at 30 degrees C occurred without additions of Fe(3+), and values of Fe(3+) never exceeded 10(2) ppm. Because of precipitate formation, an accurate assessment of the role of crystallographic orientation on the leaching of pyrite is difficult.     

Establishment of immortalized rat Kupffer cell lines

BACKGROUND: Kupffer cells have been implicated in the pathogenesis of various liver diseases. Primary cultures of Kupffer cells have a very limited life span, tend to de-differentiate and become senescent, and therefore are not suitable for cell signaling studies. AIM: To establish immortalized rat Kupffer cell lines that facilitate mechanistic studies of cell signaling and signal transduction. METHODS: Rat Kupffer cells were sub-cultured with EGF to obtain rat Kupffer Cell line 1 (RKC1), and subsequently transfected with Simian Virus 40 Large T-antigen expression vector to obtain rat Kupffer Cell line 2 (RKC2). RESULTS: RKC1 and RKC2 are similar to primary Kupffer cells as they express the molecular markers ED1, ED2, ED3, and F4/80, and upregulate TNF-alpha, IL-6, IL-1beta, Fas /FasL, and NF-kappaB, as well as TLR4 in response to LPS or pancreatic elastase. Additionally, RKC1 and RKC2 maintain phagocytic properties of latex beads and exhibit increased telomerase and stabilized p53 activity. CONCLUSION: Immortalized RKC1 and RKC2 cells maintain properties of primary Kupffer cells and can be valuable tools in evaluating the role of Kupffer cells in immune diseases and in liver-cell based drug discovery.     

The sterol‐binding activity of PATHOGENESIS‐RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein

Pathogenesis‐related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS‐RELATED 1 (PR‐1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR‐1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR‐1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol‐auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR‐1, whereas sterol‐prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR‐1 expression are particularly well protected against oomycete pathogens.     

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