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, Fluorescence, Degradation, Root, Aux/IAA
Materials and Reagents
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Procedure
Note: Expression levels of Aux/IAA proteins are typically very low, so we have modified an existing heat-shock-inducible promoter system (Gray et al., 2001) to generate high levels of a fluorescently-tagged Aux/IAA for live cell imaging in root tissues prior to hormone treatment.
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Acknowledgments
This protocol was adapted from Gray et al. (2001), and it was performed by Guseman et al. (2015) and Moss et al. (2015). This work was supported by the Paul G. Allen Family Foundation (J. L. N.), National Science Foundation (MCB-1411949 to J. L. N.) and National Institute of Health (R01-GM107084 to J. L. N.). B. L. M. received fellowship support from the National Cancer Institute of the National Institutes of Health (F32CA180514). J. M. G. was supported by the Developmental Biology Predoctoral Training Grant [T32HD007183] from the National Institute of Child Health and Human Development (NICHD). The authors thank Jodi LS Lilley for careful reading of this manuscript.
References
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