Effect of Gibberellic Acid on the Senescence of Leaf Discs of Nasturtium (Tropaeolum majus)

Abstract

Aging in leaves is characterized by a decrease in chlorophyll content and an accompanying loss of protein and ribonucleic acid. It is well known that these symptoms of senescence occur at an accelerated rate in excised mature leaves and in leaf discs floated on water. In recent years it has been established that several chemical substances retard the senescence of leaves. Among the compounds which retard senescence are the kinetins and related structures (4, 6,8, 10, 12) and auixins (3, 9). It is significant that the retardation of senescence by these substances varies from species to species. Thtus, atuxins delay senescence in Prunus leaves but are without effect on Xanthium leaves. In contrast kinetin retards leaf senescence in Xanthium btut is ineffective in Prunus (5). Recently it has been reported (2) that the gibberellins can retard leaf senescence. However, the results are again species specific. Thtus in 17 species stutdied, only Taraxacumt officiwiiile demonstrated a gibberellic acid regulation of senescence (2). During investigations in this laboratory into the senescence of leaf discs of Tropaeolunt majts it was noted that senescence coutld be prevented by gibberellic acid treatment. Thus the Nasturtiutm must be added to the species in which senescence is regutlated by gibberellin. Althouigh the mode of action of gibberellic acid in controlling senescence remains to be eluicidated, the response of leaf discs to gibberellin treatment has been established and is reported here. Nasturtiumll seed 7Tropaeolumw mnajuts (Glorious Gleam) were sown in a vermicuilite: p.at: soil mixtture (1: 1: 1) and germinated and grown in the greenhouse with a minimum temperature of 18? and natuiral illumination. Leaves of approximately 6 cm diameter were collected from these seedlings at intervals as requiired. One-cm discs were punched from the leaves, and 30 discs were floated in 20 ml of either water or gibberellic acid soluition in petri dishes. The gibberellic acid (GA) uised was the 75 % K salt stupplied by Nutritional Biochemicals Corporation and for most investigations the concentration used was 20 mg per liter. The petri dishes and contents were maintained at 220 with an illumination of 600 ft-c during a 12-hour photoperiod. At intervals 10 discs were removed from the dishes and used for chemical analyses. The leaf discs were extracted 3 times with boiling 80 % (v/v) ethanol and then grouind in 80 % (v/v) ethanol in a ground glass homogenizer. The homogenate was centriftuged at 12,000 X g for 15 minutes. The resulting supernatant fluid was combined with the initial extracts and after appropriate dilution used for the estimation of chlorophyll, alcohol-soluble carbohvdrate and alcohol-soluble nitrogen. Chlorophyll was estimated by determining the OD of the alcohol extract at 665 myu (4). Carbohydrate content of the alcohol extract was measured by the anthrone method of Yemm and Willis (13) and the alcohol-soluble nitrogen was determined by a modified Kjeldahl digestion and nesslerization ( 11). RNA, DNA and protein were extracted from the alcohol-insoluble residue according to the methods described by Osborne (4). RNA and DNA were estimated by referring OD readings of the extracts at 260 mu to similarly treated preparations of purified RNA and DNA. Protein content of the alkaline extracts was determined bv a modified Kjeldahl digestion followed by nesslerization (11). Duiring the 7-day period of study the leaf discs floating on water became yellow while those floating on GA solutions remained green. It has been found that GA was effective in preventing yellowing and chlorophyll loss in Nasturtium leaf discs at the lowest concentrations tested, 1 mg per liter. However, as a matter of routine 20 mg per liter solutions were used in all subsequtent experiments. It has been fotund that leaf discs floated in GA solutions of this concentration maintained the&r green color for at least 6 weeks. In contrast leaf discs floated on water turned yellow in 4 to 5 days. These visible differences between control and GA treated leaf discs are confirmed by data oIn chlorophyll content of the alcoholic extracts of the leaf discs. It was foulnd (table I) that over a 7-day period the chlorophyll content decreased 1 Supported by NSF grant GB-2304 and by federal funds granted to the University of Illinois Agricultuiral Experiment Station.