Effects of EGTA on the increase in Calmodulin level in early germination of radish (Raphanus sativus) seeds

EFFECTS OF EGTA ON THE INCREASE IN CALMODULIN LEVEL IN EARLY GERMINATION OF RADISH (RAPHANUS SATWUS) SEEDS. M. Cocucci and N. Negrini - Istituto di Chimica agraria, Universita di Milano, via Celoria 2 Milano. In a previous paper we have shown that the metabolic reactivation which takes place durm& germination of radish seeds (Raphanus sativus) is accompanied by an mcrease in CaImodulin (CaM) level (+450 % at 24h). The presence of a proteinaceous CaM inhibitor m ungerrninated seeds was shown and a strong reduction in its level during metabolic reactivation was re~rted. In the present study the effect of CaZ+ chelator EGTA on metabolic reactivation and increase in CaM level is reported. The presence of EGTA in the incubatIon medium inhibited germination, measured as increase in fresh weight of embryo axes, at concentrations higher than 1 mM and at 5 mM the increase in fresh weight at 24h was inhibiteiI by ca. 50%. Incubations in 1 and 5 mM EGTA reduced Ca2+ content Qf embryo axes at 24h incubation by ca. 30 and 35 % respectively. CaZ+ in the incubation medium was very low in the control. In the medIUm of the seeds incubated in the presence of EGTA the level of Ca2+ was high but the free Ca2+ was very low. Metabolic reactivation, measured as increase in RNA and DNA contents in embryo axes, was not affected by 1 mM EGTA but was slightly reduced by 5 mM EGTA; a toxic action of 5 mM EGTA was also shown by a large leakage of K+ in the incubation medium. When the seeds were incubated m the presence of EGTA and Ca2+ at the same concentration the germination and metabolic reactivation were very similar to the con!(olindi.cat~g that the e.ffect of EGTA dependend on the decrease in CaZ+availabIlity. The increase in CaM level in the soluble fraction of embryo axes incubated in the presence of 1 mM EGT A was inhibited by 50% and its specific activity was reduced in a similar way. When the soluble fraction was analyzed on a Sephadex G 75 SF column, CaM was eluted in several peaks in tlie zone at MW higher than 30 kD. In seeds incubated in the presence of EGTA there was a reduction of CaM eluted in the zone at lower MW . In such zone CaM was eluted with the inhibitor. The fact that at 1 mM EGTA in the incubation medium there was only a slight decrease in germination and metabolic reactivation even if CaM level was 50% lower suggests that Ca2+ might control the increase in CaM level which takes place in early germination and that in this same period the role of CaM could lie also in relatIon with its Ca2+ chelating capacity. The control of the activities dependent on Ca-CaM could be also in relation with the presence in the seeds of the proteinaceous CaM inhibitor.