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Live imaging of calcium spikes during double fertilization in Arabidopsis.

ABSTRACT: Ca(2+) waves and oscillation are key signalling elements during the fertilization process of animals, and are involved, for example, in egg activation. In the unique double fertilization process in flowering plants, both the egg cell and the neighbouring central cell fuse with a sperm cell each. Here we succeeded in imaging cytosolic Ca(2+) in these two cells, and in the two synergid cells that accompany the gametes during semi-in vivo double fertilization. Following pollen tube discharge and plasmogamy, the egg and central cells displayed transient Ca(2+) spikes, but not oscillations. Only the events in the egg cell correlated with the plasmogamy. In contrast, the synergid cells displayed Ca(2+) oscillations on pollen tube arrival. The two synergid cells showed distinct Ca(2+) dynamics depending on their respective roles in tube reception. These Ca(2+) dynamics in the female gametophyte seem to represent highly specific signatures that coordinate successful double fertilization in the flowering plants.

SUBMITTER: Hamamura Y

PROVIDER: S-EPMC4143913 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Live imaging of calcium spikes during double fertilization in Arabidopsis.

Hamamura Yuki Y Nishimaki Moe M Takeuchi Hidenori H Geitmann Anja A Kurihara Daisuke D Higashiyama Tetsuya T

Nature communications 20140822

Ca(2+) waves and oscillation are key signalling elements during the fertilization process of animals, and are involved, for example, in egg activation. In the unique double fertilization process in flowering plants, both the egg cell and the neighbouring central cell fuse with a sperm cell each. Here we succeeded in imaging cytosolic Ca(2+) in these two cells, and in the two synergid cells that accompany the gametes during semi-in vivo double fertilization. Following pollen tube discharge and pl ...[more]

PMID: 25146889

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Project description:During double fertilization in angiosperms, two male gametes (sperm cells), are released from a pollen tube into the receptive region between two female gametes; the egg cell and the central cell of the ovule. The sperm cells fertilize the egg cell and the central cell in a one-to-one manner to yield a zygote and an endosperm, respectively. The one-to-one distribution of the sperm cells to the two female gametes is strictly regulated, possibly via communication among the four gametes. Polyspermy block is the mechanism by which fertilized female gametes prevent fertilization by a secondary sperm cell, and has been suggested to operate in the egg cell rather than the central cell. However, whether the central cell also has the ability to avoid polyspermy during double fertilization remains unclear. Here, we assessed the one-to-one fertilization mechanism of the central cell by laser irradiation of the female gametes and live cell imaging of the fertilization process in Arabidopsis thaliana. We successfully disrupted an egg cell within the ovules by irradiation using a femtosecond pulse laser. In the egg-disrupted ovules, the central cell predominantly showed single fertilization by one sperm cell, suggesting that neither the egg cell nor its fusion with one sperm cell is necessary for one-to-one fertilization (i.e., monospermy) of the central cell. In addition, using tetraspore mutants possessing multiple sperm cell pairs in one pollen, we demonstrated that normal double fertilization was observed even when excess sperm cells were released into the receptive region between the female gametes. In ovules accepting four sperm cells, the egg cell never fused with more than one sperm cell, whereas half of the central cells fused with more than one sperm cell (i.e., polyspermy) even 1 h later. Our results suggest that the central cell can block polyspermy during double fertilization, although the central cell is more permissive to polyspermy than the egg cell. The potential contribution of polyspermy block by the central cell is discussed in terms of how it is involved in the one-to-one distribution of the sperm cells to two distinct female gametes.

Project description:Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program.Significance statementAxon degeneration resulting from trauma or neurodegenerative disease can cause devastating deficits in neural function. Understanding the molecular and cellular events that execute axon degeneration is essential for developing treatments to address these conditions. Calcium is known to contribute to axon degeneration, but its temporal requirements in this process have been unclear. Live calcium imaging in severed zebrafish neurons and temporally controlled pharmacological treatments in both zebrafish and cultured mouse sensory neurons revealed that axonal calcium influx late in the degeneration process regulates axon fragmentation. These findings suggest that temporal considerations will be crucial for developing treatments for diseases associated with axon degeneration.

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Live imaging of calcium spikes during double fertilization in Arabidopsis.

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Live imaging of calcium spikes during double fertilization in Arabidopsis.

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