Imaging Ca2+ Dynamics in Wild-Type and NADPH Oxidase-Deficient Mutant Pollen Tubes with Yellow Cameleon and Confocal Laser Scanning Microscopy
While cytosolic calcium (Ca2+) plays a central role in a myriad of signaling pathways as a secondary messenger, how dynamic changes of cytosolic calcium relate to cell growth control remains poorly understood. The engineering and continuous improvements of genetically encoded calcium sensors such as the Yellow Cameleon (YC) sensors combined with advances in microscopy have allowed imaging with great resolution of the spatiotemporal characteristics of cytosolic [Ca2+]cyt in individual cells. An exciting new step consists therefore in cautiously studying calcium dynamics in mutant backgrounds that display disturbed cellular growth behavior to further enhance our understanding on growth-related processes. Here, we describe methods to perform imaging of [Ca2+]cyt dynamics in growing Arabidopsis thaliana wild-type and NADPH-oxidase deficient rbohH rbohJ pollen tubes stably expressing YC3.6 using confocal laser scanning microscopy. We also present different ways to extract meaningful qualitative and quantitative information about calcium dynamics during growth.
Key wordsArabidopsis thaliana Pollen tube Ca2+ YC3.6 Ratio-imaging Cell growth NADPH oxidase Cell wall integrity
We are grateful to Megumi Iwano (Osaka University, Japan) for gifting us seeds of the pACT1-YC3.60 line. We thank all members of Martin Hülskamp’s group (University of Cologne, Germany) for sharing their facilities and CEPLAS for access to the Leica SP8 confocal microscope. This work was supported by the University of Cologne, the Deutsche Forschungsgemeinschaft Grant BO 4470/1-1, and a grant from the University of Cologne Centre of Excellence in Plant Sciences to A.B.D.
Two minute long time course of a steady growing WT PT with FRET, CFP, ratio and DIC channels. Scale bar is 10 μm (AVI 69125 kb)
Two minute long time course of an unsteady growing rbohH-3 rbohJ-3 PT with FRET, CFP, ratio and DIC channels. Scale bar is 10 μm (AVI 69125 kb)
Two minute long time course of a bursting rbohH-3 rbohJ-3 PT with FRET, CFP, ratio and DIC channels. Scale bar is 10 μm (AVI 69125 kb)
- 10.Kong SK, Lee CY (1995) The use of fura 2 for measurement of free calcium concentration. Biochemist 23:97–98Google Scholar