Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals

Abstract

Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca2+ in the presence of ATP. The specific activity of ATP-dependent uptake is .apprx.20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca2+ stimulation of ATP hydrolysis was not discernible above the high background of Ca2+-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca2+ concentration, suggesting that Ca2+ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca2+ at half maximal velocity of uptake (S0.5) was 12.5 micromolar and the Hill coefficient (nH) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca2+, although this may reflect that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca2+ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca2+ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca2+ into microsomal vesicles declined by .apprx.70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of sensecence. There were no significant changes during this period in S0.5 or nH, but Vmax for ATP-dependent Ca2+ uptake decreased by .apprx.40%. A similar decline in ATP-dependent uptake of Ca2+ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene.