The Levels of Soluble Nucleotides in Wheat Aleurone Tissue

Abstract

The content of soluble nucleotides in aleurone layers isolated from mature wheat (Triticum aestivum var. Olympic) grain was investigated. The most abundant nucleotides were adenosine triphosphate, uridine triphosphate, and uridine diphosphoglucose. Smaller amounts of guanosine triphosphate, cytidine triphosphate, adenosine diphosphate, and nicotinamide adenine dinucleotide were also identified. The levels of some of these nucleotides were increased after incubation of the tissue under certain conditions. Nucleotide levels were measured at intervals during incubation of aleurone layers in water. The changes observed are discussed in relation to a response by the tissue to wounding.Gibberellic acid applied to the aleurone layer of mature cereal grain in the absence of the embryo stimulates the activity and secretion of a number of hydrolytic enzymes (10,20,22). A lag phase of some 6 to 8 hr occurs between the addition of GA3 and the first detectable increase in enzyme activity (9, 12).Investigators of the mode of action of GA. have paid close attention to its effect on nucleic acid metabolism (8,25,32 were bisected transversely; and the embryo halves were discarded.The following solvents were redistilled before use: triethylamine, isobutyric acid, ethanol, diethyl ether (over FeSO4 and CaO) and 1-propanol (over KOH). Water was distilled twice and deionized. All other chemicals and solvents used were of reagent grade.All equipment and water was sterilized before use by autoclaving at 121 C and 6.8 kg pressure for 20 min. A sterilizing solution for the half-seeds was prepared by suspending 5 g CaOCl in 100 ml water, shaking the suspension for 10 min, and then filtering. The half-seeds were soaked in the filtrate for 2 hr, rinsed 10 times in water, and then allowed to imbibe water for 24 hr at 30 C.After imbibition, the half-seeds were aseptically transferred to a sterile glass jar; then a weighted polyethylene bottle (sterilized by immersion in 70% ethanol) was inserted in the jar. The jar was capped and rotated at 50 rpm for 30 min (23) with periodic additions of water. The suspension was filtered, and the tissue was returned to the jar and rolled for a further 7 to 8 min. After further filtration and thorough rinsing, the tissue was drained on absorbent paper and weighed out for use. Phillips and Paleg (23) found that bacterial and fungal colonies were almost completely absent even 36 hr after isolation of the tissue. Since most incubations used here were 24 hr or less, no further precautions were taken.The technique used to extract and separate the soluble nucleotides was modified from procedures reported by Cole and Ross (11), Isherwood and Barrett (13), and Jenner (15). Treated tissue was transferred to a cold room, and all operations, unless otherwise stated, were carried out at 4 C. The tissue was first rinsed in ice-cold water and drained on absorbent paper, and then it was disintegrated with an Ultra-Turrax in 5% (w/v) trichloroacetic acid containing 0.15% (w/v) 8-hydroxyquinoline. Each extract was ...

Adenine Nucleotide Content of Corn Roots as Affected by Injury and Subsequent Washing

“…Similar changes in adenine nucleotides (and other metabolites) during washing or aging of excised plant tissue have been reported by other investigators (7,21,23,25). The phenomenon must reflect some important metabolic change in the cells which is linked to wounding or other injury, and in corn roots may have a role in inducing the developmental phase.…”

Lysosomal nature of hormonally induced enzymes in wheat aleurone cells

“…Enzyme assays a-Amylase activity was determined by a modification (Collins et al, 1972a,b) of the method of Shuster & Gifford (1962), after inactivation of ,-amylase activity by heating at 70°C for 20min (Paleg, 1960). Amylose (BDH Chemicals Ltd., Poole, Dorset, U.K.) was used as substrate.…”

The Metabolism of Soluble Nucleotides in Wheat Aleurone Layers Treated with Gibberellic Acid

“…Thus, a study of the effect of GA3 on the metabolism of these compounds could be expected to show aspects of the action of the hormone not readily detectable by other methods. In a previous paper (7), techniques were described for measuring the free nucleotides of wheat aleurone layers. This tissue responds to GA3 in a fashion apparently identical to that of barley aleurone and, in contrast to the latter, can be easily prepared in sufficient quantity (22) After treatment, both the surrounding medium and the tissue were assayed for a-amylase activity.…”