The involvement of Ca²⁺ gradients, Ca²⁺ fluxes, and CaM kinase II in polarization and germination of Silvetia compressa zygotes

Abstract

Previous work has shown that distinct Ca²⁺ gradients precede and predict the loci of germination of the zygotes of the brown alga, Silvetia compressa (J. Agardh) E. Serrão, T.O. Cho, S.M. Boo et Brawley, that are polarized by unilateral blue light. We show here that dark-grown S. compressa zygotes also form cytosolic Ca²⁺ gradients prior to germination and then germinate from the site of elevated Ca²⁺. In no case did germination occur without a prior formation of a Ca²⁺ gradient. Using the self-referencing Ca²⁺-selective probe, we measured highly localized influx of Ca²⁺ during photopolarization, indicating that extracellular stores supply at least some of the Ca²⁺ needed to construct a gradient. Finally, we find that germination was inhibited by a bath-applied inhibitor of calcium/calmodulin-dependent kinase II (CaM kinase II), KN-93 (but not by its inactive analog, KN-92), and by an injected inhibitory peptide for the kinase. KN-93 did not interfere with the photopolarization of the zygotes, consistent with the view that calmodulin is not involved in the initial response to light. The KN-93 results indicate that the requirement for active CaM kinase II for germination ends about 2 h before overt germination. We conclude that Ca²⁺ gradients, generated in part by localized calcium entry from the seawater, are an essential part of the process of polarity development and expression in these cells, regardless of the nature of the external cue that directs the orientation of the axis. Calmodulin and CaM kinase II are involved in interpreting (but not in establishing) the calcium gradient, allowing germination to occur at the site of elevated calcium, but CaM kinase II appears not to be involved in the initiation of germination.