Published: Vol 6, Iss 2, Jan 20, 2016 DOI: 10.21769/BioProtoc.1712 Views: 11784
Reviewed by: Arsalan DaudiKaisa KajalaAnonymous reviewer(s)
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Abstract
Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.
Keywords: PlantMaterials and Reagents
Equipment
Procedure
Representative data
Expression of the MYC2p-GUS reporter in seedlings is normally confined to the roots, basal part of the hypocotyl and leaf primordia (Figure 3A; Gasperini et al., 2015) but single cotyledon wounding induces further activation in above ground tissues, including cotyledons and the upper hypocotyl (Figure 3B). Repetitive cotyledon wounding causes a reduction in root length due to decreased meristem cell number associated with reduced expression of the cell cycle gene CYCB1;1 (Gasperini et al., 2015), as visualized by a lower CYCB1;1p-GUS reporter activity in the root meristem of treated seedlings (Figure 3C-D).
Figure 3. Examples of promoter-GUS activities following single and repetitive cotyledon wounding. MYC2p-GUS reporter activity in control (A) and single cotyledon wounded seedlings (B). The GUS enzyme converts X-Gluc into a blue colored product revealing sites of transcriptional activity. The orange asterisk indicates the wounding site. CYCB1; 1p-GUS reporter expression in the root meristem of the control (C) and seedling subjected to repetitive cotyledon wounding (D). Scale bars = 0.5 mm (A, B); 50 μm (C, D).
Notes
Recipes
Acknowledgments
The single wounding protocol was developed by Acosta et al. (2013) and the repetitive wounding protocol by Gasperini et al. (2015). This work was supported by the Swiss National Science Foundation grant 31003A-138235 to E.E.F. We thank A. Chételat, A. Kurenda and C. T. Nguyen for photography and video assistance.
References
Article Information
Copyright
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Gasperini, D., Acosta, I. F. and Farmer, E. E. (2016). Cotyledon Wounding of Arabidopsis Seedlings. Bio-protocol 6(2): e1712. DOI: 10.21769/BioProtoc.1712.
Category
Plant Science > Plant physiology > Abiotic stress
Plant Science > Plant physiology > Tissue analysis
Plant Science > Plant immunity > Perception and signaling
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