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

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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.