Use of fluorescent reporters to analyse dynamic and spatial responses to mechanical wounding

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Larrieu, A. | Nguyen, T.H. | Champion, Antony

Edité par CCSD ; Humana Press -

Mechanical wounding of plant tissues triggers many different responses (Savatin DV, Gramegna G, ModestiV, Front Plant Sci 5:470, 2014). These are primarily mediated by the plant hormone Jasmonic AcidIsoleucine (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 withoutapparent damage to the seedling, affecting downstream analyses (Larrieu A, Champion A, Legrand J, NatCommun 6:6043, 2015). In this chapter, we describe how to overcome this technical limitation to monitorany fluorescent reporter or dye in response to wounding, using any type of fluorescent or confocal (invertedor upright, laser scanning or spinning disc) microscopes. Pharmacological or wound treatments caneasily be performed and responses monitored over long periods of time. We further describe a simplemethod 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.

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