Robert L. Shewfelt

Does the alternative pathway ameliorate chilling injury in sensitive plant tissues

Free radical processes have been observed in senescence and several membrane-associated disorders of plants including chilling, freezing, and desiccation injuries. The mitochondria of plant tissues exposed to low temperatures, and other abiotic and biotic stresses, produce superoxide and/or hydrogen peroxide when electron transport through the cytochrome pathway is impaired due to the energy state of the cell or to stress-induced physical changes in the membrane components. The superoxide and/or hydrogen peroxide produced can diffuse throughout the cell causing peroxidation of membrane lipids which results in membrane disruption, increased permeability and metabolic disturbances, and eventually the visible symptoms of chilling injury. The alternative pathway of electron transport in the mitochondria, which is induced by low temperatures in some plant tissues, can mediate these degradative processes by reducing the level of superoxide generated by the mitochondria.

Peroxidation of isolated microsomal membranes from nongreen plant tissue

Summary Lipid peroxidation has been linked to membrane-associated disorders in plant tissue, but proposed mechanisms differ on whether phospholipid hydrolysis in membranes accelerates or inhibits peroxidation. Isolated microsomal fractions from cowpea seeds and cauliflower florets were challenged by hydrolytic (PLA 2 ) and oxidative (FeCl 2 -ADP or FeCl 3 -ascorbate) conditions simultaneously or in sequence to determine the effect of hydrolysis on peroxidation in plant membranes. Cauliflower microsomes were more susceptible to peroxidative challenge than cowpea microsomes by an order of magnitude. Lipid peroxidation of cowpea microsomes was stimulated by phospholipid hydrolysis even prior to challenge by prooxidants. A slight, but consistent inhibition of lipid peroxidation was observed in cauliflower microsomes preincubated wtih PLA 2 . The results suggest that other factors in plant membranes are more important in the progress of lipid peroxidation than the presence or absence of free fatty acids.

Isolation of plasma membrane from Capsicum annum fruit tissue: prevention of acid phosphatase contamination

Isolation of highly purified plasma membrane fractions from outer pericarp tissue of mature-green bell pepper (Capsicum annum) fruit was achieved using the two-phase polymer partitioning method. This system was optimized by adjusting the partition pH and noting the effect on various cell fraction markers. The modified system [5.8% (ww) PEG (MW 3380), 5.8% (ww) Dextran T-500, 20 mM NaCl, 5 mM KPi, pH 7.3] dramatically decreased thylakoid and Golgi apparatus membrane contamination. Use of normal homogenization medium resulted in acid phosphatase contamination, as evidenced by 30–40% inhibition of the apparent ATPase activity by 0.1 mM molybdate. A modified homogenization medium containing 0.3 M sucrose, 250 mM Tris-HCl (pH 8.5), 25 mM EDTA, 5 mM EGTA, 2.5 mM dithiothreitol (DTT), and 1 mM PMSF, and alternatively a microsome washing step efficiently reduced acid phosphatase contamination from 37% of the total plasma membrane ATPase activity to 8 and 12%, respectively. Two-phase partitioning of senescing fruit tissue provides a tool for studying the role of the plasma membrane in degradative changes observed during postharvest storage.