Synthesis of Isocitrate Lyase in Sunflower Cotyledons during the Transition in Cotyledonary Microbody Function

Abstract

Density-labeling with 10 nillimolar K"NO3/70% 2H20 has been used to investigate isocitrate lyase synthesis during greening of sunflower (Helianthus auas L.) cotyledons when the glyoxysomal enzyme activities sharply decline and the transition in cotyledonary microbody function occurs. A density shift of 0.0054 (kilograms per liter) was obtained for the profile of isocitrate lyase activity in the CsCI gradient with respect to the 'H20 control. Quantitative evaluation of the density-labeling data indicates that about 50% of the isocitrate lyase activity present towards the end of the transition stage in microbody function is due to enzyme molecules newly synthesized during this stage. Fat-storing cotyledons of certain seeds, including those of the sunflower, become functional as leaves in the normal course of germination. During early stages of development the stored fat is mobilized, and if the cotyledons emerge from the soil and gain exposure to light they develop into photosynthetic organs. During this changeover in cotyledonary metabolism, the microbody population of the mesophyll cells loses its glyoxysomal activities and acquires the enzymic characteristics of leaf peroxisomes. It is still a matter of discussion as to how this transition in microbody function occurs (1, 7, 8). One reason for the current difficulty in interpreting published biochemical data as unequivocal evidence either for or against any one of the discussed hypotheses, is our ignorance of the existence and, if so, extent of turnover of glyox-ysomes during the transition stage in microbody function. Results which demonstrate the occurrence of isocitrate lyase synthesis during normal greening of fatty cotyledons are presented in this paper. These results were obtained by density-labeling the glyox-ysomal marker enzyme and quantitatively evaluating the data obtained. MATERIALS AND METHODS Plant Material and Standard Growth Conditions. Achenes of sunflower (Helianthus annuus L., var. Spanners Allzweck) were soaked 12-14 h and then germinated in moist Vermiculite at 30 C. After 2.5 days ofgrowth in darkness the seedlings were exposed to continuous light. The counting of days of germination was begun with the planting of the soaked achenes. Density-Labeling. For density-labeling, a solution of 10 miM K'5N03 in 70% 2H20 was used. The procedure for introducing the isotope solution into the cotyledons of embryos and of 2.5-day-old, dark-grown seedlings, as well as the growth conditions for labeled embryos and seedlings, were those described by Betsche 'This research was supported by the Deutsche Forschungsgemeinschaft.