Calmodulin levels in radish (Raphanus sativus L.) seeds germinating at low calcium availability induced by EGTA treatments

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Abstract
Incubation of radish (Raphanus sativus L.) seeds in the presence of 1 or Smol m−3 Ca‐EGTA, which increased Ca2+ activity in the incubation medium (c. 0.24 or 0.37 mol m−3 at 24 h with respect to c. 0.13 mol m−3 in the control), did not affect germination, the restoration of K+ net influx, the increase in DNA and RNA levels or protein synthesis. Incubation in 1 mol m−3 Na‐EGTA, which reduced Ca2+ activity in the incubation medium (20 mmol m−3 at 24 h), decreased the total Ca2+ level in embryo axes (‐21%), but only slightly inhibited the increase in fresh weight without affecting the restoration of K+ net influx, the increase in DNA and RNA levels or protein synthesis. In the presence of 5 mol m−3 Na‐EGTA (Ca2+ activity in the incubation medium was 0.6 mmol m−3), the decrease in the total Ca2+ level was greater (c. ‐27%) and the increases in fresh weight, DNA and RNA were inhibited by about 50, 39 and 40%, respectively. These results indicate that increased Ca2+ availability does not affect germination and suggest that the effect of Na‐EGTA, at least up to 5 mol m−3, is a result of an induction of Ca2+ deficiency. The amount and specific activity of calmodulin (CaM) present in the soluble fraction (100 000g) of radish embryo axes greatly increased during the first 24 h of incubation (c. 5‐fold and 7‐fold, respectively). This increase was very similar in the Ca‐EGTA‐treated seeds but was inhibited (c. ‐38%) by 1 mol m−3 Na‐EGTA, even if the increases in DNA and RNA levels and protein synthesis were not significantly reduced. The lower amount of CaM after 24 h of incubation in 1 mol m−3 Na‐EGTA (c. ‐30%) was due to a reduction in the fraction of CaM bound to a proteinaceous CaM inhibitor present in radish seeds [M. Cocucci & N. Negrini (1988) Plant Physiology 88, 910–914] and not involved in the metabolic reactivation of the seed. These results suggest that the level of CaM is controlled by Ca2+ availability and that the CaM inhibitor has a role in controlling the amount of Ca‐CaM available for the Ca‐CaM‐dependent enzymes.