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 Ca(2+) in the presence of ATP. The specific activity of ATP-dependent uptake is approximately 20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca(2+) stimulation of ATP hydrolysis was not discernible above the high background of Ca(2+)-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca(2+) concentration, suggesting that Ca(2+) serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca(2+) at half maximal velocity of uptake (S(0.5)) was 12.5 micromolar and the Hill coefficient (n(H)) 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 Ca(2+), although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca(2+) 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 Ca(2+) transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca(2+) into microsomal vesicles declined by approximately 70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S(0.5) or n(H), but V(max) for ATP-dependent Ca(2+) uptake decreased by approximately 40%. A similar decline in ATP-dependent uptake of Ca(2+) into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene.