Changes in the Levels of Calmodulin and of a Calmodulin Inhibitor in the Early Phases of Radish (Raphanus sativus L.) Seed Germination

Abstract

An inhibitor of Ca2_-calmodulin (Cam)-dependent brain phosphodiesterase was present in the soluble fraction of embryo axes from ungerminated radish (Raphanus sativus L.) seeds. This inhibitor is a Ca2+-dependent, Cam-binding protein; in fact: (a) its effect was strongly reduced by treatment with proteases; (b) the inhibition was counteracted by Cam but not by Ca2 ; (c) on gel filtration in the presence of Ca2+, Cam co-chromatographed with the inhibitor. The inhibitor is heat stable and positively charged at pH 7.5. During early phases of germination, the fresh weight and the levels of DNA and RNA of embryo axes increased, the level of the inhibitor decreased, and the level of Cam increased. Abscisic acid (ABA) inhibited germination, the decrease of inhibitor, and the increase of Cam. Fusicoccin (FC) stimulated the increase in fresh weight but not the increase in the RNA and DNA levels; in this condition, the inhibitor level decreased and the increase in Cam level was higher than in the control. In the presence of both ABA and FC, there was an increase in fresh weight not accompanied by an increase in DNA and RNA levels; Cam increased and, on a fresh weight basis, reached the value of the control. These results indicate that the Ca2+-Cam system was activated in early germination of radish seeds by an increase in Cam and a decrease in the inhibitor levels, that FC, probably through the activation of membrane functions, increased Cam level, and that the ABA inhibition on germination was not mediated by the Ca2+-Cam system. (12), to the maturation of oocytes of Xenopus (6), and to DNA synthesis and mitosis (27). In plants a relationship between Cam level and cell growth is suggested by the fact that higher levels (17-fold) of Cam were found in the root apex (meristem) with respect to the zone of cell elongation (1) and by results on seed germination. Cam level increases during early phases of germination; this increase is reduced when germination is inhibited by ABA, and Cam level appears to be a limiting factor for the Ca2+-Cam-dependent activities (8).An inhibitor of Ca2+-Cam-dependent PDE is present in radish (Raphanus sativus L.) seeds. This inhibitor could be involved in the regulation of Ca2+-Cam-dependent activities and, consequently, of seed germination (8).Seed germination is also characterized by the reactivation of membrane functions (4, 9). FC, a toxin able to stimulate proton extrusion and K+ absorption and to increase the negative electric transmembrane potential (19), stimulates germination, while ABA inhibits germination and the reactivation of membrane functions. FC reverses, in early germination phases, the inhibition of ABA on membrane functions and germination (4).The present paper deals with the evidence that the inhibitor, present in radish seeds, is a Cam-binding protein and reports the changes in Cam and inhibitor levels during early germination phases of radish embryos when germination was stimulated by FC or inhibited by ABA. The role of Cam and of the inhibitor in germination is d...

Purification and Sequencing of Radish Seed Calmodulin Antagonists Phosphorylated by Calcium-Dependent Protein Kinase

“…2). This polypeptide pattern is similar to that found for a calmodulin antagonist preparation from radish (Cocucci and Negrini, 1988). In view of this heterogeneity, the RCA preparation was subjected to denaturation by heating in the presence of 3 M guanidinium HCl and 0.1 M 2-mercaptoethanol and subsequent reverse-phase HPLC on a Cs matrix, as described in "Materials and Methods."…”

“…The RCAs are very basic proteins that inhibit calmodulin function and are similar in these properties and in molecular size to calmodulin antagonist proteins previously partially purified from radish seeds (Cocucci and Negrini, 1988). These calmodulin-inhibiting proteins disappear during germination, and ABA inhibits both germination and the decrease in the levels of the inhibitor proteins (Cocucci and Negrini, 1988).…”

“…Because a calmodulin antagonist protein preparation had been previously resolved from radish seeds (Cocucci and Negrini, 1988), we examined the fractions eluting from the SP5PW column for calmodulin antagonist activity. Calmodulin antagonist activity was measured by determining inhibition of chicken gizzard Ca2+-ca1modulin-dependent MLCK activity, employing as a protein substrate a synthetic peptide substrate (KKRAARATSNVFA-NH2) of this enzyme (the sequence of this peptide relating to the phosphorylateable region of myosin light chains).…”

“…These calmodulin-inhibiting proteins disappear during germination, and ABA inhibits both germination and the decrease in the levels of the inhibitor proteins (Cocucci and Negrini, 1988). Germination is associated with an increase in calmodulin and a decrease in calmodulin inhibitory activity (Cocucci and Negrini, 1988).…”

The 58-Kilodalton Calmodulin-Binding Glutamate Decarboxylase Is a Ubiquitous Protein in Petunia Organs and Its Expression Is Developmentally Regulated

“…Moreover, a significant decrease in these antagonists and a simultaneous increase in CaM levels were observed during early phases of germination, implying that CaM-regulated processes are activated (Cocucci and Negrini, 1988). It is conceivable that the 58-kD CaM-binding GAD in petunia is one of the enzymes that are activated by Ca 2+ /CaM in the course of germination.…”