Aleurone

Abstract: Gibberellic acid enhances the synthesis of α-amylase in isolated aleurone layers of barley-seeds (Hordeum vulgare var. Himalaya). In the presence of 20 mm calcium chloride the amount of enzyme obtained from isolated aleurone layers is quantitatively comparable to that of the half-seeds used in earlier studies. After a lag period of 6 to 8 hours enzyme is produced at a linear rate. Gibberellic acid does not merely trigger α-amylase synthesis, but it is continuously required during the period of enzyme formation. Enzyme synthesis is inhibited by inhibitors of protein and RNA synthesis. Small amounts of actinomycin D differentially inhibit enzyme release and enzyme synthesis suggesting 2 distinct processes. Gibberellic acid similarly enhances the formation of ribonuclease which increases linearly over a 48 hour period. During the first 24 hours the enzyme is retained by the aleurone cells and this is followed by a rapid release of ribonuclease during the next 24 hour period. The capacity to release the enzyme is generated between 20 and 28 hours after the addition of the hormone. Ribonuclease formation is inhibited by inhibitors of protein and RNA synthesis. These inhibitors also prevent the formation of the release mechanism if added at the appropriate moment.

Abstract: Germination of radish (Raphanus sativus cv Eterna) seeds can be inhibited by far-red light (high-irradiance reaction of phytochrome) or abscisic acid (ABA). Gibberellic acid (GA3) restores full germination under far-red light. This experimental system was used to investigate the release of reactive oxygen intermediates (ROI) by seed coats and embryos during germination, utilizing the apoplastic oxidation of 2′,7′-dichlorofluorescin to fluorescent 2′,7′-dichlorofluorescein as an in vivo assay. Germination in darkness is accompanied by a steep rise in ROI release originating from the seed coat (living aleurone layer) as well as the embryo. At the same time as the inhibition of germination, far-red light and ABA inhibit ROI release in both seed parts and GA3 reverses this inhibition when initiating germination under far-red light. During the later stage of germination the seed coat also releases peroxidase with a time course affected by far-red light, ABA, and GA3. The participation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in ROI metabolism was demonstrated with specific in vivo assays. ROI production by germinating seeds represents an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens.

Abstract: Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of α-amylase. α-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.