Abstract
The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH.
Keywords: Auxin signaling, Cell wall pH, Cell elongation, Hypocotyl, Live-cell imaging
Background
Phytohormone auxin induces rapid growth in Arabidopsis thaliana hypocotyls. This process requires the TIR1/AFB-Aux/IAA auxin co-receptor. Auxin promotes the binding of TIR1/AFB and Aux/IAA, which leads to ubiquitination and degradation of the latter, and results in transcription of auxin-responsive genes. This protocol focuses on measuring the growth, auxin signaling and cell wall acidification in Arabidopsis thaliana etiolated hypocotyls. This protocol is based on the previous work of Schenck et al., 2010, Takahashi et al., 2012, Fraas et al., 2014 and Spartz et al., 2014; but unlike the published work, we describe the procedures that enable measuring a larger spectrum of processes occurring during growth of hypocotyls; from the macroscopically visible organ elongation, cell wall pH monitored by confocal microscopy to the real-time nuclear auxin signaling visualized by luciferase bioluminescence.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
During all steps where the hypocotyls are manipulated (cut and transferred to new plates) it is crucial to be extremely gentle with the tissue, not squeeze it but rather scoop it using sharp forceps. The tissue needs to be protected from drying; plates must be kept closed whenever possible to prevent excessive evaporation.
Recipes
Acknowledgments
This protocol was adapted from Fendrych et al., 2016. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 665385, and Austrian Science Fund (FWF) [M 2128-B21]. The authors declare no conflict of interests.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.