Comparative detection of calcium fluctuations in single female sex cells of tobacco to distinguish calcium signals triggered by in vitro fertilization.
Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca(2+)-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca(2+) in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca(2+) signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 microM Fluo-3 for 30 min at 30 degrees C. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca(2+)](cyt) of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.
Peng
Xiong-Bo
XB
Key Laboratory of Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Sun
Meng-Xiang
MX
Yang
Hong-Yuan
HY
D000814:Aniline Compounds / Q000737:chemistry; D020013:Calcium Signaling / Q000502:physiology*; D005306:Fertilization / Q000502:physiology*; D035264:Flowers / Q000166:cytology* / Q000378:metabolism*; D018613:Microscopy, Confocal; D014026:Nicotiana / Q000166:cytology* / Q000378:metabolism*; D014966:Xanthenes / Q000737:chemistry
D000814:Aniline Compounds; D014966:Xanthenes; C059715:Fluo-3