Abstract
Plant-hormone-initiated signaling pathways are extremely vital for plant growth, differentiation, development, and adaptation to environmental stresses. Hormonal perception by receptors induces downstream signal transduction mechanisms that lead to plant responses. However, conventional techniques—such as genetics, biochemistry, and physiology methods—that are applied to elucidate these signaling pathways can only provide qualitative or ensemble-averaged quantitative results, and the intrinsic molecular mechanisms remain unclear. The present study developed novel methodologies based on in vitro single-molecule fluorescence assays to elucidate the complete and detailed mechanisms of plant hormone signal transduction pathways. The proposed methods are based on multicolor total internal reflection fluorescence microscopy and a flow cell model for gas environment control. The methods validate the effectiveness of single-molecule approaches for the extraction of abundant information, including oligomerization, specific gas dependence, and the interaction kinetics of different components.
Highlights
Plant hormones are naturally occurring organic substances that influence physiological processes at low concentrations (Davies, 1995)
General Considerations In the employed total internal reflection fluorescence microscopy (TIRFM)-based single-molecule fluorescence technique, if the target proteins are moving freely in the buffer, the dwell time of the target protein flowing through the focal plane is shorter than common camera frame rates
The interaction readout can be in the format of colocalization single-molecule spectroscopy (CoSMoS) (Friedman and Gelles, 2012), FRET (Förster, 1948; Clegg, 1992), or BiFC (Bimolecular fluorescence complementation) (Hu et al, 2002)
Summary
Plant hormones are naturally occurring organic substances that influence physiological processes at low concentrations (Davies, 1995). In addition to the five classical phytohormones, namely auxins, abscisic acid (ABA), cytokinin (CK), gibberellins (GAs), and ethylene, additional compounds have been recognized as hormones, including brassinosteroids (BRs), jasmonate, salicylic acid, nitric oxide, and strigolactones (Santner and Estelle, 2009). These hormones govern every aspect of plant biological processes, including developmental processes, signaling networks, and responses to biotic and abiotic stresses (Bari and Jones, 2009). ABA modulates stomatal closure, inhibits shoot growth, and induces storage protein synthesis in seeds (Davies, 2010). Different hormones regulate distinct members of protein families; they may share components and modulate similar processes (Nemhauser et al, 2006)
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