In this paper, we report direct measurement of an influx of extracellular Ca 2؉ induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca 2؉ -selective vibrating probe. Ca 2؉ influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca 2؉ influx in the vicinity of the sperm entry site with a delay of 1.8 ؎ 0.6 sec. The Ca 2؉ influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 m⅐sec ؊1 . Once established, Ca 2؉ influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol⅐cm ؊2 maize ͉ channels ͉ vibrating ion probe F ertilization in higher plants is an extremely complex process, involving the interaction of several cells and tissues and a double fusion inside the embryo sac. In maize, in vitro fertilization (IVF) procedures allow the in vitro manipulation and study of the two cytological fusions that lead to a full seed: the sperm-egg cell fusion that initiates the development of the embryo (1-3) and the sperm-central cell fusion that leads to the formation of the endosperm (4). Attention has been paid to the changes triggered by sperm-egg fusion at the cellular and molecular levels (reviewed in refs. 5 and 6). In particular, a transient elevation of cytosolic calcium concentration ( c [Ca 2ϩ ]) has been observed to be triggered after sperm-egg fusion in a medium containing 5mM CaCl 2 (7). Because increasing or decreasing external Ca 2ϩ changed significantly the rate of spermegg fusion (3), the extracellular Ca 2ϩ dependence of this calcium rise was not investigated in this study.One question not yet addressed concerns the origin of this calcium rise. Data from animal systems indicate that calcium can be mobilized from internal stores and͞or arise from an influx of extracellular Ca 2ϩ during fertilization, depending on the species investigated (8). In brown algae, a calcium influx is believed to be induced by gamete fusion and seems to play a direct role in egg activation (9, 10). We do not know yet whether this is the case for flowering plants.The Ca 2ϩ selective vibrating probe (for review, see refs. 11 and 12) developed a decade ago by Kühtreiber and Jaffe (13) is a powerful and strictly noninvasive method for measuring minute extracellular calcium gradients with spatial and temporal resolutions of a few microns͞seconds. The calcium-vibrating electrode records externally the net flux of calcium across the plasma membrane of a single cell by measuring potential differences between two points perpendicular to the plasma membrane and by subsequently converting the microvolt signal to flux data and current densities. Application of this technique to pollen tubes (14-16) and roots (17)(18)(19)(20) indicates the potential of the vibrating probe for recording and analyzi...

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 fusion 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 controversial. In higher plants, in vitro fertilization (IVF) has enabled the description of a rise in [Ca2+]Cyt (ref. 4) that is sufficient for activation, and of a Ca2+ influx that spreads as a wavefront from the fusion site 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.