Abstract
The most direct technique for studying calcium, which is an essential element for pollen tube growth, is Ca2+ imaging. Because membranes are relatively impermeable, the loading of fluorescent Ca2+ probes into plant cells is a challenging task. Thus, we have developed a new method of loading fluo-4 acetoxymethyl ester into cells that uses a cell lysis solution to improve the introduction of this fluorescent dye into pollen tubes. Using this method, the loading times were reduced to 15 min. Furthermore, loading did not have to be performed at low (4°C) temperatures and was successful at room temperature, and pluronic F-127 was not required, which would theoretically allow for the loading of an unlimited number of cells. Moreover, the method can also be used to fluorescently stain root hairs.
Highlights
Pollen tube growth is crucial for the delivery of sperm cells to the ovule during angiosperm reproduction
The pollen tubes that were not exposed to cell lysis solution showed no fluorescence at a loading time of 30 min, the cell walls and membranes did produce a weak fluorescence (Fig 1I and 1J)
These results suggest that the cell lysis solution can promote fluo-4/A combination of tetra (AM) loading into pollen tubes
Summary
Pollen tube growth is crucial for the delivery of sperm cells to the ovule during angiosperm reproduction. The role of intracellular Ca2+ can only be investigated by monitoring its concentration dynamics in plant cells with both spatial and temporal accuracy [3]. Because critical measurements of [Ca2+]i are essential for assessing transduction pathways and downstream events in tip-growing cells, the applied imaging method is important. Encoded calcium sensors can efficiently measure calcium concentrations in cellular organelles, especially in in vitro or in vivo single cells. Calcium sensors can be used in transformation and cell biology to study sexual organs that are accessible and visualized and handled [4], such as those of Nicotiana tabacum [5] and Arabidopsis thaliana [2]. In certain plants the construction of a stable transgenic system is difficult; the use of small, fluorescent, calcium-sensing molecules has been enormously beneficial for PLOS ONE | DOI:10.1371/journal.pone.0152320 April 7, 2016
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