活体成像的热激诱导系统测定植株的Aux/IAA降解率

Jessica M. Guseman; Jennifer L. Nemhauser; Britney L. Moss

doi:10.21769/BioProtoc.1881

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Peer-reviewed

A Live-imaging, Heat Shock-inducible System to Measure Aux/IAA Degradation Rates in Planta

活体成像的热激诱导系统测定植株的Aux/IAA降解率

JG Jessica M. Guseman

JN Jennifer L. Nemhauser

BM Britney L. Moss email

发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1881 浏览次数: 10011

评审: Arsalan DaudiEunsook ParkStefanie Rosa

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Sep 2015

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Abstract

An emerging theme in biology is the importance of cellular signaling dynamics. In addition to monitoring changes in absolute abundance of signaling molecules, many signal transduction pathways are sensitive to changes in temporal properties of signaling components (Purvis and Lahav, 2013). The phytohormone auxin regulates myriad processes in plant development. Many of these require the nuclear auxin signaling pathway, in which degradation of the Aux/IAA repressor proteins allows for transcription of auxin-responsive genes (Korasick et al., 2015). Using a heterologous yeast system, we found that Aux/IAAs exhibit a range of auxin-induced degradation rates when co-expressed in isolation with F-box proteins (Havens et al., 2012). Subsequent studies connecting signaling dynamics to plant growth and development confirmed that Aux/IAAs show similar differences in plants (Guseman et al., 2015; Moss et al., 2015). Here, we describe in detail the use of a heat-shock-inducible fluorescence degradation system to capture Aux/IAA degradation in real time in live plant roots. By employing this method, we were able to obtain high Aux/IAA expression and avoid the dampening long term effects of turnover, feedback and silencing. Degradation was dependent on the presence of an Aux/IAA degron and rates increased in response to exogenous auxin.

Keywords: Auxin (生长素)

Materials and Reagents

1,000 μl pipet tip (Thermo Fisher Scientific, Fisherbrand^TM, catalog number: 02-681-4 )  
50 ml conical centrifuge tube (Thermo Fisher Scientific, Fisherbrand^TM, catalog number: 06-443-20 )  
1.5 ml microcentrifuge tubes (Thermo Fisher Scientific, Fisherbrand^TM, catalog number: 05-408-129 )  
Square (100 x 100 x 15 mm) Petri plates (Thermo Fisher Scientific, Fisherbrand^TM, catalog number: 08-757-11A )
Round (35 mm) Petri plates (VWR International, catalog number: 10799-192 )  
Micropore tape 1530-0 (3M, catalog number: 70200412230 )  
Aluminum foil
Razor blades
Rectangular (24 x 50 mm) microscopy coverslips (Thermo Fisher Scientific, catalog number: 12-544E )
Labeling tape  
> 4 ml glass perfume spray bottles (available in craft stores)  
Arabidopsis thaliana Columbia-0 (Col) seeds transformed with heat-shock constructs (Guseman et al., 2015; Moss et al., 2015) 
Bacto Agar (BD, catalog number: 214010 )
Linsmaier-Skoog Media (Caisson Laboratories, catalog number: LSP03-1LT ) (see Note 1)
Triton X-100 (GE Healthcare, catalog number: US22686 )  
Indole-3-acetic acid (IAA) (bioWORLD, catalog number: 705490 )
Kanamycin (Thermo Fisher Scientific, catalog number: BP906-5 )
95% ethanol
Seed sterilization solution (see Recipes)
0.5x Linsmaier-Skoog (LS) liquid media (see Recipes)
Sterile plating agar solution (see Recipes)
0.5x LS media + 0.8% agar (see Recipes)
Cover slip media (0.5x LS Media + 1.2% agar) (see Recipes)
IAA stock solution (5 mM) (see Recipes)

Equipment

Slide warmer (Thermo Fisher Scientific, model: 11-474-470 )
Microscope (Leica Biosystems, model: DMI 3000B ) fitted with a Lumencor SOLA light source and YFP filter cube
Leica long working 40x HCX PL FLUOTAR objective (see Note 2)
Leica camera (Leica Microsystems, model: DFC345 FX ) 
Forceps (Electron Microscopy Sciences, model: Dumont Tweezers Style 2A )
Timer

Software

Leica LAS AF version 2.6.0 for image acquisition
Fiji/ImageJ software for image analysis  
GraphPad Prism6 software package for graphing and statistical analysis

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Guseman, J. M., Nemhauser, J. L. and Moss, B. L. (2016). A Live-imaging, Heat Shock-inducible System to Measure Aux/IAA Degradation Rates in Planta. Bio-protocol 6(15): e1881. DOI: 10.21769/BioProtoc.1881.
Moss, B. L., Mao, H., Guseman, J. M., Hinds, T. R., Hellmuth, A., Kovenock, M., Noorassa, A., Lanctot, A., Villalobos, L. I., Zheng, N. and Nemhauser, J. L. (2015). Rate motifs tune auxin/indole-3-acetic acid degradation dynamics. Plant Physiol 169(1): 803-813.