Differential contribution of cytoplasmic Ca²⁺ and Ca²⁺ influx to gamete fusion and egg activation in maize

Abstract

In multicellular organisms, gamete fusion triggers a set of events, collectively known as egg activation, that leads to the development of a new individual. Every species that has been studied shows at least one rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_Cyt) after gamete fusion¹ which is believed to be involved in activation. Yet the source and regulation of this Ca²⁺ signal and the way it is transduced inside the zygote are controversial¹. In higher plants, in vitro fertilization (IVF)^2,3 has enabled the description of a rise in [Ca²⁺]_Cyt (ref. 4) that is sufficient for activation⁵, and of a Ca²⁺ influx that spreads as a wavefront from the fusion site⁵ The relationship between these two responses is unknown. Using a new combination of methods that simultaneously monitor the extracellular flux with a Ca²⁺-vibrating probe, and [Ca²⁺]_Cyt by widefield imaging, we directly determined that the Ca²⁺ influx precedes the [Ca²⁺]_Cyt elevation by 40–120 s. In addition, results from experiments using the Ca²⁺-channel inhibitor gadolinium (Gd³⁺) suggest that the Ca²⁺ influx may be necessary for sperm incorporation. We also present evidence for a putative sperm-dependent Gd³⁺-insensitive localized Ca²⁺ influx confined to the fusion point.