Use of Fluorescent Reporters to Analyse Dynamic and Spatial Responses to Mechanical Wounding

Mechanical wounding of plant tissues triggers many different responses (Savatin DV, Gramegna G, Modesti V, Front Plant Sci 5:470, 2014). These are primarily mediated by the plant hormone Jasmonic Acid Isoleucine (JA-Ile). Recently, a fluorescent biosensor for JA-Ile showed that sample preparation (i.e., handling of samples) for fluorescent microscopy very often triggers wound response, even without apparent damage to the seedling, affecting downstream analyses (Larrieu A, Champion A, Legrand J, Nat Commun 6:6043, 2015). In this chapter, we describe how to overcome this technical limitation to monitor any fluorescent reporter or dye in response to wounding, using any type of fluorescent or confocal (inverted or upright, laser scanning or spinning disc) microscopes. Pharmacological or wound treatments can easily be performed and responses monitored over long periods of time. We further describe a simple method to extract and analyse quantitative data from confocal images using the open source software Fiji (Fiji Is Just ImageJ (Schindelin J, Arganda-Carreras I, Frise E, Nat Methods 9:676-682, 2012)) and OpenOffice.