Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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

发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1881 浏览次数: 10983

评审: Arsalan DaudiEunsook ParkStefanie Rosa

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

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

Advertisement

“科研可重复性与创新”系列网络研讨会
Special Issue on Protocols for Organoid Research
Technologies for RNA Detection
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

地衣藻<em>Trebouxia</em>鞭毛阶段的周期性活细胞观察简易实验方案

地衣藻Trebouxia鞭毛阶段的周期性活细胞观察简易实验方案

Enrico Boccato [...] Mauro Tretiach

2026年01月20日 416 阅读

利用活细胞成像检测蓝猪耳柱头中的触碰诱导钙动态变化

利用活细胞成像检测蓝猪耳柱头中的触碰诱导钙动态变化

Xuan Zhou [...] Shihao Su

2026年05月05日 334 阅读

褐藻细胞的四维成像

褐藻细胞的四维成像

Marie Zilliox and Bénédicte Charrier

2026年07月05日 267 阅读

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) search Fluorescence (荧光) search Degradation (降解) search Root (根) search Aux/IAA (Aux/IAA) search

Materials and Reagents

  1. 1,000 μl pipet tip (Thermo Fisher Scientific, FisherbrandTM, catalog number: 02-681-4 ) 

  2. 50 ml conical centrifuge tube (Thermo Fisher Scientific, FisherbrandTM, catalog number: 06-443-20 ) 

  3. 1.5 ml microcentrifuge tubes (Thermo Fisher Scientific, FisherbrandTM, catalog number: 05-408-129 ) 

  4. Square (100 x 100 x 15 mm) Petri plates (Thermo Fisher Scientific, FisherbrandTM, catalog number: 08-757-11A )
  5. Round (35 mm) Petri plates (VWR International, catalog number: 10799-192 ) 

  6. Micropore tape 1530-0 (3M, catalog number: 70200412230 ) 

  7. Aluminum foil
  8. Razor blades
  9. Rectangular (24 x 50 mm) microscopy coverslips (Thermo Fisher Scientific, catalog number: 12-544E )
  10. Labeling tape 

  11. > 4 ml glass perfume spray bottles (available in craft stores) 

  12. Arabidopsis thaliana Columbia-0 (Col) seeds transformed with heat-shock constructs (Guseman et al., 2015; Moss et al., 2015)

  13. Bacto Agar (BD, catalog number: 214010 )
  14. Linsmaier-Skoog Media (Caisson Laboratories, catalog number: LSP03-1LT ) (see Note 1)
  15. Triton X-100 (GE Healthcare, catalog number: US22686 ) 

  16. Indole-3-acetic acid (IAA) (bioWORLD, catalog number: 705490 )
  17. Kanamycin (Thermo Fisher Scientific, catalog number: BP906-5 )
  18. 95% ethanol
  19. Seed sterilization solution (see Recipes)
  20. 0.5x Linsmaier-Skoog (LS) liquid media (see Recipes)
  21. Sterile plating agar solution (see Recipes)
  22. 0.5x LS media + 0.8% agar (see Recipes)
  23. Cover slip media (0.5x LS Media + 1.2% agar) (see Recipes)
  24. IAA stock solution (5 mM) (see Recipes)

Equipment

  1. Slide warmer (Thermo Fisher Scientific, model: 11-474-470 )
  2. Microscope (Leica Biosystems, model: DMI 3000B ) fitted with a Lumencor SOLA light source and YFP filter cube
  3. Leica long working 40x HCX PL FLUOTAR objective (see Note 2)
  4. Leica camera (Leica Microsystems, model: DFC345 FX )

  5. Forceps (Electron Microscopy Sciences, model: Dumont Tweezers Style 2A )
  6. Timer

Software

  1. Leica LAS AF version 2.6.0 for image acquisition
  2. Fiji/ImageJ software for image analysis 

  3. GraphPad Prism6 software package for graphing and statistical analysis

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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

  1. 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.
  2. 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.

    3. ris ris Download Citation in RIS Format

分类

细胞生物学 > 细胞成像 > 活细胞成像

植物科学 > 植物生物化学 > 植物激素

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件